Nucleic acid analysis by random mixtures of non-overlapping fragments

ABSTRACT

The invention provides methods and kits for ordering sequence information derived from one or more target polynucleotides. In one aspect, one or more tiers or levels of fragmentation and aliquoting are generated, after which sequence information is obtained from fragments in a final level or tier. Each fragment in such final tier is from a particular aliquot, which, in turn, is from a particular aliquot of a prior tier, and so on. For every fragment of an aliquot in the final tier, the aliquots from which it was derived at every prior tier is known, or can be discerned. Thus, identical sequences from overlapping fragments from different aliquots can be distinguished and grouped as being derived from the same or different fragments from prior tiers. When the fragments in the final tier are sequenced, overlapping sequence regions of fragments in different aliquots are used to register the fragments so that non-overlapping regions are ordered. In one aspect, this process is carried out in a hierarchical fashion until the one or more target polynucleotides are characterized, e.g. by their nucleic acid sequences, or by an ordering of sequence segments, or by an ordering of single nucleotide polymorphisms (SNPs), or the like.

This application claims priority from U.S. provisional applications Ser.No. 60/776,415 filed 24 Feb. 2006, Ser. No. 60/725,116 filed 7 Oct.2005, and Ser. No. 60/690,771 filed 15 Jun. 2005, each of which ishereby incorporated by reference in its entirety.

GOVERNMENT INTERESTS

This invention was made with government support under grant No. 1 U01AI057315-01 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for determining nucleotidesequences and/or marker maps of large nucleic acids, such as genomes orparts of genomes, and more particularly, to methods for reconstructingsequences of large nucleic acids from sequences of many fragmentsthereof.

BACKGROUND

The goal set by National Human Genome Research Institute to promote thedevelopment of technology for sequencing mammalian-sized genomes forunder $1000. was a dramatic acknowledgement of the tremendous value thatnucleic acid sequence data has in virtually every area of the lifesciences, Collins et al (2003), Nature, 422: 835-847. This challenge hasspurred interest in many different sequencing approaches as alternativeto, or complements of, Sanger-based sequencing, which has been thework-horse sequencing technology for the last two decades, e.g.Margulies et al (2005), Nature, 437: 376-380; Shendure et al (2005),Science, 309: 1728-1732; Kartalov et al, Nucleic Acids Research, 32:2873-2879 (2004); Mitra et al, Anal. Biochem., 320: 55-65 (2003);Metzker (2005), Genome Research, 15: 1767-1776; Shendure et al (2004),Nature Reviews Genetics, 5: 335-344; Balasubramanian et al, U.S. Pat.No. 6,787,308; and the like. A common attribute of many of these newapproaches is the acquisition of sequence information from many shortrandomly selected fragments in a highly parallel manner. Massive amountsof sequence information are generated that must be processed toreconstruct the sequence of the larger polynucleotide from which thefragments originated. Unfortunately such processing presents asignificant hurdle to many genome sequencing projects because of thewell-known difficulties of reconstructing long polynucleotides fromshort sequences, e.g. Drmanac et al, Advances in Biochem. Engineering,77: 75-101 (2002).

Another difficulty faced by current and developing sequence technologiesarises from the diploid nature of many organisms of interest. That is,the cells of all mammals and many other organisms of interest containtwo copies of every genomic sequence and the pair of such sequencesdiffer from one another by a small but significant degree due to naturalallelic variation, mutations, and the like. Thus, when diploid genomesare reconstructed from shorter sequences, it is very difficult todetermine which difference should be allocated to which sequence of thepair. A similar difficulty arises when sequencing populations oforganisms as well, e.g. Tringe et al (2005), Nature Reviews Genetics, 6:805-814. In the latter case, there are mixtures of pathogens (forexample, HIV or other viruses) where complete viral or bacterial strainor haplotype determination is critical for identifying an emergingresistant organism or man-modified organism mixed with non-virulentnatural strains.

In view of the above, it would be highly useful, particularly to manysequencing technologies under development, to have available a techniquethat would allow the generation of additional information about thelocation of short sequence reads in a genome.

SUMMARY OF THE INVENTION

The invention provides methods and kits for determining nucleotidesequences and/or marker maps of one or more target polynucleotides. Inone aspect, the invention provides a method of characterizing nucleotidesequences of one or more target polynucleotides comprising the steps of:(a) forming a plurality of tiers of mixtures that comprise a hierarchyof nested fragments of the one or more target polynucleotides, eachmixture of each prior tier being divided into a number of mixtures in asubsequent tier, at least one tier having mixtures with substantiallynon-overlapping fragments, and the plurality of tiers having a finaltier wherein mixtures of prior tiers can be identified for each fragmentof each mixture of the final tier; (b) determining sequence informationfrom at least a portion of one or more fragments of each mixture in thefinal tier; and (c) providing complete or partial nucleotide sequencesof the one or more target polynucleotides by ordering the sequenceinformation from the final tier of mixtures, wherein such orderingdepends on the identity of at least one mixture of at least one tierfrom which a fragment is derived that gives rise to a portion of suchsequence information.

In another aspect, the invention provides a method of characterizingnucleotide sequences of one or more target polynucleotides comprisingthe steps of: (a) fragmenting the one or more target polynucleotidespresent in a predetermined coverage amount to form a populationcontaining overlapping first-sized fragments each having an averagelength substantially less than those of the target polynucleotides; (b)forming a number of separate mixtures from the population of first-sizedfragments, such number being selected such that substantially everyfirst-sized fragment in a separate mixture is non-overlapping with everyother first-sized fragment of the same mixture, and such that themixture of origin of each such first-sized fragment can be identified;(c) determining sequence information from at least a portion of one ormore first-sized fragments of each mixture; and (d) providing completeor partial nucleotide sequences of the one or more targetpolynucleotides by ordering the sequence information from the separatemixtures, wherein such ordering depends on the mixture of origin of atleast a portion of the sequence information.

In still another aspect, the invention provides a method of preparingfor sequence analysis one or more target polynucleotides present in apredetermined coverage amount, the method comprising the followingsteps: (i) fragmenting the one or more target polynucleotides to form apopulation containing overlapping first-sized fragments each having anaverage length substantially less than those of the targetpolynucleotides; (ii) aliquoting the population of first-sized fragmentsinto a number of separate mixtures, such number being selected such thatsubstantially every first-sized fragment in a separate mixture isnon-overlapping with every other first-sized fragment of the sameseparate mixture; and (iii) attaching an oligonucleotide tag to eachfirst-sized fragment in each separate mixture so that theoligonucleotide tag identifies the separate mixture of the first-sizedfragment.

The invention further includes kits for implementing the methods of theinvention. In one aspect, such kits comprise reagents and/or mechanicalappliances for generating fragments of one or more targetpolynucleotides. In another aspect, such kits comprise reagents forattaching oligonucleotide tags to fragments generated from one or moretarget polynucleotides and divided into separate mixtures in accordancewith methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate different aspects of the invention.

FIGS. 2A-2B illustrate methods of circularizing genomic DNA fragmentsfor generating concatemers of polynucleotide analytes.

FIGS. 3A-3C illustrate a high-throughput sequencing method that may beused with the invention.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The invention provides methods and kits for ordering sequenceinformation derived from one or more target polynucleotides. In oneaspect, one or more tiers or levels of fragmentation and aliquoting aregenerated after which sequence information is obtained from fragments ina final level or tier. Each fragment in such a final tier is from aparticular aliquot, which, in turn, is from a particular aliquot of aprior level or tier. For every fragment of an aliquot in the final tier,the aliquots from which it was derived at every prior level is known.Thus, identical sequences from overlapping fragments from differentaliquots can be distinguished and grouped as being derived from the sameor different fragments from prior levels. When the fragments in thefinal tier are sequenced, overlapping sequence regions of fragments indifferent aliquots are used to register the different fragments so thatnon-overlapping regions are ordered. In one aspect, this process iscarried out in a hierarchical fashion until the one or more targetpolynucleotides are characterized, e.g. by their nucleic acid sequences,or by an ordering of sequence segments, or by an ordering of singlenucleotide polymorphisms (SNPs), or the like. In another aspect,fragments at each tier are tagged with an oligonucleotide tag toidentify the tier and aliquot of the separate mixture, after whichfragments of the final tier may be mixed (either to form a singlemixture or multiple mixtures) and analyzed together, for example, by wayof a high-throughput sequencing device, e.g. Margulies et al (2005),Nature, 437: 376-380; Shendure et al (2005), Science, 309: 1728-1732.The results of such sequencing is the acquisition of sequenceinformation of final fragments coupled with identification of one ormore oligonucleotide tags, which, in turn, identify fragments from priortiers that a final fragment is derived from. Sequences of the tags,since they are selected from a known set, may also be used to improvebase calling, or be used as a quality control measure for sequencing.Such tags may also mark the end sequences of longer fragments beforesubsequent fragmenting and may be used to guide sequence or mapassembly. In one aspect, oligonucleotide tag may be added to fragmentsby replicating fragment using tagged primers; that is, primers that havea fragment binding portion, which may be a random sequence, e.g. 6 to 18bases in length, and a portion (usually a 5′ portion) that does not bindto fragments that contains an oligonucleotide tag.

Oligonucleotide tags are identified by their nucleotide sequences. Suchidentification may be accomplished as part of sequencing final tierfragments (that is, the nucleotide sequence determined includes thesequence of an oligonucleotide tag as well as the nucleotide sequence ofa portion of a final tier fragments). Alternatively, sequences ofoligonucleotide tags may be identified by hybridization probes, e.g. ona microarray.

A common feature in all applications of this invention (genomesequencing, SNP or other marker mapping or cDNA analysis) is aliquotingnucleic acid sample such that sequences of predetermined type ofrelatedness (overlapped fragment, fragments with high similarity,homologous chromosomes, messengers transcribed from the same gene) occurmostly once e.g. as a single molecule per aliquot.

FIG. 1A provides an illustration of one aspect of the invention. Ndiploid genomes (100) are shown aligned prior to fragmentation belowscale (102) that illustrates positions of fragments within the genomesby a number between 0 and 1. (That is, target polynucleotides (100) arepresent in a coverage amount of “2N.”) After fragmentation (104) eachgenome is broken into multiple overlapping fragments that co-exist inone mixture (108), where fragments of one strand of top-most genome(106) (solid lines) are label by their position in the genome. Fragmentsof mixture (108) are then divided into a number of aliquots 1 through Ksuch that the likelihood of any one aliquot receiving overlappingfragments is small, e.g. less than one percent. (Many or all aliquotsmay have one or a few pairs of overlapped fragments, but any givensegment of a polynucleotide or a pair or a group of multiplerelated/homologous polynucleotides is represented in most of thealiquots (e.g., >90%) by a single thus non-overlapping fragment. Ifthere are two overlapping fragments from a polynucleotide in one aliquotthey appear as one longer fragment. Only non-overlapped segments of twooverlapped fragments one from each parental chromosome would providehaplotype information.) In one aspect, minimal overlapping of fragmentsinsures that the fragments can be unambiguously sequenced withoutconfounding affects caused by the presence of substantially overlappingfragments that may contain small differences, such as may be foundwhenever heterozygous parental strands are present or whenever strandsof a mixed-strain population of microbes are present. Usually, afterfragmentation either before or after formation of separate mixtures, thefragments are replicated in order increase the amount of target materialfor analysis. In one aspect, fragments are replicated after the havebeen separated into separate mixtures using a conventional replicationtechnique that does not bias the amounts of different sequencesamplified. In one aspect, such first tier fragments are furtherfragmented (112) within their respective aliquots to form a second tierof fragments (114). Usually, prior to fragmentation (112), fragments ofeach aliquot may be replicated using a conventional DNA replicationprocess, such as whole genome amplification using random primers and ahighly processive DNA polymerase with strand displacement activity, e.g.U.S. Pat. No. 6,617,137, which is incorporated herein by reference.Preferably the replication method does not bias the relative amounts ofeach fragment. It is understood that replication by some methods willreduce the average fragment length. Second tier fragments (114) are thenanalyzed by any number of analytical assays, as noted above. Preferably,second tier fragments (114) are analyzed by a highly parallel DNAsequencing method, such as the one described more fully below, or likemethod. As noted above, the steps of fragmenting and aliquoting may becarried out multiple times, as illustrated in FIG. 1B, to generatemultiple levels or tiers of fragments. There, target polynucleotide(120) present in coverage amount 2N is fragmented (122) to form mixture(124), which is then separated (126) into aliquots 1 through K. Asabove, K is selected to minimize the probability of having overlappingfragments within the same aliquot. Fragments of each aliquot arereplicated then further fragmented (128) to form a second tier or levelof fragments (130). Fragments from each aliquot of the second tier maythen be further divided (132) into aliquots (134) and again replicatedand fragmented to form a third tier or level of fragments (136). In FIG.1B, third tier aliquots (1 through S) and fragments are shown only forfragments derived from aliquot 2 of the first level.

In one aspect of the invention, only a single level of fragmenting iscarried out. A method for characterizing nucleotide sequences of one ormore polynucleotides that exemplifies this aspect is carried out withthe following steps: (i) fragmenting the one or more targetpolynucleotides present in a predetermined coverage amount to form apopulation containing overlapping first-sized fragments each having anaverage length substantially less than those of the targetpolynucleotides; (ii) forming a number of separate mixtures from thepopulation of first-sized fragments, such number being selected suchthat substantially every first-sized fragment in a separate mixture isnon-overlapping with every other first-sized fragment of the samemixture and such that the mixture of origin of each such first-sizedfragment is determinable; (iii) determining sequence information from atleast a portion of one or more first-sized fragments of each mixture;and (iv) providing complete or partial nucleotide sequences of the oneor more target polynucleotides by ordering the sequence information fromthe separate mixtures, wherein such ordering depends on the mixture oforigin of at least a portion of the sequence information. As with othermethods of the invention, mixtures from which a fragment is derived canbe determined by attaching an oligonucleotide tag to each fragment in amixture, as discussed more fully below.

In one aspect, sequence information from fragments is in the form ofsequence reads. That is, sequence information comprises a nucleotidesequence of a portion of a fragment, frequently an end of a fragment.The length of such sequence reads depends on the sequencing techniqueused to analyze the fragments. In one aspect, sequence reads havelengths in the range of from 12 to 600 bases; and in another aspect,sequence reads have lengths in the range of from 20 to 100 bases; or inthe range of from 20 to 50 bases. For each separate mixture, a number ofsequence reads are acquired so that sequences of the fragments of themixtures are substantially covered, i.e. represented in the numbersequence reads. Clearly, the larger the number of sequence reads thegreater the likelihood that the sequences are covered by a given amountor percentage. In one aspect, substantially covered means that at least30 percent of such sequences are covered; or at least 50 percentcovered; or at least 66 percent covered; or at least 75 percent covered.

In one aspect, sequence reads are determined from concatemers offragments, as described more fully below, using the following steps: (i)generating for each separate mixture a plurality of target concatemersfrom the first-sized fragments, each target concatemer comprisingmultiple copies of a portion of a first-sized fragment and eachplurality of target concatemers including a number of such portions thatsubstantially covers the first-sized fragment; (ii) forming for each ofthe separate mixtures a random array of target concatemers fixed to asurface at a density such that at least a majority of the targetconcatemers are optically resolvable; and (iii) generating a number ofsequence reads from target concatemers of each of said separatemixtures, such sequence reads each having a length substantially lessthan those of said first-sized fragments, and each of such numbers ofsequence reads being selected such that the sequences reads of each saidseparate mixture substantially covers said first-sized fragmentstherein. In regard to selecting a plurality of concatemers so that theone or more target polynucleotides are covered, the degree of coveragedepends in part on the type of analysis being undertaken. For example,for determining the complete nucleotides sequences of the one or moretarget polynucleotides, the degree of coverage usually at least twotime, or more usually at least five times, the total length of the oneor more target polynucleotides, e.g. Waterman, Introduction toComputational Biology: Maps, Sequences and Genomes (Chapman & Hall/CRC,1995); Lander et al, Genomics, 2: 231-239 (1988); and the like. Forpartial sequence analysis, e.g. ordering methylation-rich regions, alesser degree of coverage may be sufficient. In regard to selecting anumber of sequence reads to substantially cover fragments in a mixture,usually the degree of coverage is less than 100 percent, as noted above.

In another aspect, sequence information from fragments is in the form ofthe presence or absence of given polymorphisms, such as singlenucleotide polymorphisms (SNPs), which may be measured by a variety ofmethods, e.g. Syvanen (2005), Nature Genetics Supplement, 37: S5-S10;Gunderson et al (2005), Nature Genetics, 37: 549-554; Fan et al (2003),Cold Spring Harbor Symposia on Quantitative Biology, LXVIII: 69-78; andU.S. Pat. Nos. 4,883,750; 6,858,412; 5,871,921; 6,355,431; and the like,which are incorporated herein by reference. In one aspect, sequenceinformation comprises the determination of the presence or absence ineach separate mixture of at least 5 SNPs, or at least 10 SNPs, or atleast 30 SNPs, or at least 50 SNPs, or at least 100 SNPs.

Implementing the invention involves several design choices within thepurview of those of ordinary skill. Such design choices includeselecting the following parameters: fragment sizes at each level,coverage amount of target polynucleotides, number of aliquots at eachlevel, number of levels, type of sequence information to obtain, thedegree and method of fragment replication at each level, and the like.Typically, after extraction and/or purification by conventional means,DNA or RNA is fragmented enzymatically or mechanically. Preferably,target polynucleotides are randomly fragmented so that overlappingfragments are produced. At each level after fragmentation, the reactionmixture containing the fragments is divided into multiple separatemixtures. This may be done by dividing the mixture into aliquots. Or,alternatively, portions of the reaction mixture may be separated intoseparate mixtures such that not all of the original reaction mixture isused. Usually, the mixture is divided into a number of equal sizedaliquots. A number of aliquots (or separate mixtures) is selected sothat there is only a minimal probability that the fragments in suchmixtures overlap (or maximally tolerable to reduce cost of handlinglarge number of aliquots). In one aspect, a number of separate mixturesis selected so that the probability of overlapping fragments is lessthan 10%; in another aspect, less than 5%; in another aspect, less than1%; in still another aspect, less than 0.1%; and in another aspect, lessthan 0.01%. In another aspect, a number of separate mixtures is selectedso that at least sixty percent of such separate mixtures contain onlynon-overlapping fragments; or in another aspect, a number of separatemixtures is selected so that at least eighty percent of such separatemixtures contain only non-overlapping fragments; or in another aspect, anumber of separate mixtures is selected so that at least ninety percentof such separate mixtures contain only non-overlapping fragments.Clearly, the number of separate mixtures or aliquots to achieve a givenprobability depends on the coverage amount of target polynucleotide usedin the method. “Coverage amount” means a factor times the amount ofnucleic acid equivalent to one copy of the target polynucleotides. Forexample, 1 ng of human genomic DNA is equivalent to about 300 copies ofa haploid human genome. Thus, a coverage amount of 300 for a humanhaploid genome as a target polynucleotide is 1 ng. Aliquoting orcreating separate mixtures may be done by conventional pipetting intoconventional laboratory vessels, such as, tubes or all or some of thewells of one or more 96-well, or 384-well, or 1536-well plates. In oneaspect, coverage amounts are in the range of from 2 to 50, or in therange of from 5 to 40, or in the range of from 5 to 30, or in the rangeof from 5 to 20. In one aspect, target polynucleotide DNA is randomlyfragmented using conventional methods including, but not limited to,sonication, passage through capillaries, dispersion of DNA solution intofine drops, treatment with DNase I, treatment with endonuclease, taggedPCR amplification, and the like, e.g. Dienenger (1983) Anal. Biochem.,129: 216-223; Schriefer et al (1990), Nucleic Acids Research, 18: 7455;Anderson et al (1996) Anal. Biochem., 236: 107-113; Anderson et al(1981) Nucleic Acids Research, 9: 3015-3027; Fitzgerald et al (1992)Nucleic Acids Research, 20: 3753-3762; Grothues et al, Nucleic AcidsResearch, 21: 1321-1322; Zheleznaya et al (1999), Biochemistry (Moscow)64: 373-378; and the like. It is understood that in some instancestarget polynucleotides are fragmented in the course of conventionalextractions methods, so that, in particular, an initial step offragmenting may simply result from conventional extraction and handlingof target polynucleotides, e.g. mammalian genomic DNA, or the like.Average fragment size may be selected in each of these methods byroutine parameter choices. In one aspect, four levels of fragmentationare implemented wherein fragments of the first fragmentation, i.e.first-sized fragments, are in a range of from 100-300 kilobases (kb),fragments of the second fragmentation, i.e. second-sized fragments, arein a range of from 10-30 kilobases (kb), fragments of the thirdfragmentation, i.e. third-sized fragments, are in a range of from 1-3kilobases (kb), and fragments of the fourth or final fragmentation, i.e.final-sized fragments, are in a range of from 50-600 bases. In anotheraspect, average fragment sizes are selected relative to the length ofthe target polynucleotides. Thus, for example, selecting averagefragment sizes substantially less than those of fragments from the priortier means selecting an average size less than one third the size of theaverage size of fragments of the prior tier; or in another aspect,selecting an average size less than one tenth the size of the averagesize of fragments of the prior tier; or in another aspect, selecting anaverage size less than one thirtieth the size of the average size offragments of the prior tier; or in another aspect, selecting an averagesize less than one hundredth the size of the average size of fragmentsof the prior tier; or in another aspect, selecting an average size lessthan one thousandth the size of the average size of fragments of theprior tier.

In one aspect, having a subset of aliquots with much fewer fragmentsthan the others is useful for sequence reconstruction. That is, insteadof dividing a mixture of fragments into equal volume separate mixtures,which on average would result in equal numbers of fragments in eachseparate mixture, a mixture of (for example) first-sized fragments maybe divided into separate mixtures that include subsets with fewerfragments. For human genome, for example, if a mixture is divided intoonly 384 separate mixtures, a subset of 96 such mixtures may have onlyabout four hundred 100 kb fragments each and the other 3×96 separatemixtures may have about four thousand 100 kb fragments each. The lowcomplexity separate mixtures (˜40 Mb of genomic DNA) may be used tosimplify sequence assembly especially for de-novo analysis. Sequencesfrom such low complexity separate mixtures may be used to find all othersequences in high complexity aliquots that overlap with them and thusform low complexity subsets from the large set of about 6 billiongenerated sequence reads. The low complexity sets allows more efficientfinal sequence assembly. If more separate mixtures are formed, such as1536 (especially efficient by tagging second-sized fragments), then 384or 2×384 aliquots may have only fifty to one hundred 100 kb fragmentsrepresenting only 5-10 Mb of genomic DNA.

In another aspect, having an initial population of long fragments thatare highly overlapped provides the best conditions to assemble parentalchromosomes. For 20× and 100 kb fragments, the neighboring fragmentswill have 95 kb overlap, on average. 20× coverage will also assure thatin large majority of cases the overlap of consecutive fragments is atleast 10 kb (e.g. having about 10 SNPs that differentiate two parentalchromosomes).

As mentioned above, in one aspect, the invention provides a method ofcharacterizing nucleotide sequences of one or more targetpolynucleotides comprising the steps of: (a) forming one or more tiersof mixtures that comprise a hierarchy of nested fragments of the one ormore target polynucleotides, each mixture of each prior tier beingdivided into a number of mixtures in a subsequent tier so that at leastone tier has mixtures with substantially non-overlapping fragments, andthe one or more tiers having a final tier wherein mixtures of priortiers can be identified for each fragment of each mixture of the finaltier; (b) determining sequence information from at least a portion ofone or more fragments of each mixture in the final tier; and (c)providing complete or partial nucleotide sequences of the one or moretarget polynucleotides by ordering the sequence information from thefinal tier of mixtures, wherein such ordering depends on the identity ofat least one mixture of at least one tier from which a fragment isderived that gives rise to a portion of such sequence information. Inone aspect, the number of tiers is one; and in another aspect, thenumber of tiers is two; and in further aspects, the number of tiers maybe a plurality greater than two. For example, the plurality may bethree, or it may be four. As used herein, the term “hierarchy of nestedfragments” means levels (or equivalently, tiers) of fragmentationwherein the levels are related in that fragments of each successivelevel are derived from the fragments from an immediately prior level.Moreover, fragments derived from an immediately prior level have averagelengths that are substantially less than those of the fragments fromwhich they are derived; hence, they are nested in that sense. The natureand origin of the one or more target polynucleotides may vary widely.The one or more target polynucleotides may comprise mRNAs or cDNAs, orthey may comprise whole genomes or fragments of genomes. One or moretarget polynucleotides may be one or more bacterial genomes, or one ormore fungal genomes, or one or more mammalian genomes, or one or moreplant genomes, or fragments of any of the preceding. In another aspect,one or more target polynucleotides may comprise one or more strains orspecies of bacterial or viral genomes. In still another aspect, one ormore target polynucleotides may comprise one or more genomes or genomefragments from a community of organisms, such as enteric bacterial,vaginal microorganisms, or the like.

As mention above, sequence information may be derived from manydifferent types of analysis, such as, SNP measurements, nucleotidesequence determination, determination of methylated bases, restrictionsites, DNA binding sites, and the like. In one aspect, sequenceinformation comprises nucleotide sequence determination of at least aportion of substantially every final fragment. Such information can beobtained by any of the available sequencing techniques, but those thatare amenable to convenient highly parallel sequencing of many hundredsor thousands, or hundreds of thousands, or millions of fragmentssimultaneously are preferred. Such techniques may provide nucleotidesequences of varying lengths, i.e. they may have differing “readlengths.” In one aspect, read lengths of from 10 to 500 are obtained; orread lengths of from 20 to 100 are obtained. In other aspects, sequenceinformation is obtained from multiple levels in a single operation, sothat for example, a number of fragments from a first tier may besequenced with a first technique that provides read lengths in a rangeof from 100 to 1000 nucleotides, whereas another number of fragments(usually greater than the first number) are sequenced with a secondtechnique that provides read lengths in the range of from 20 to 100nucleotides. After such sequence information is obtained, assemblingsuch information to reconstruct the sequences of fragments within analiquot or of fragments or contigs within the sample is well-known, asevidenced by the following exemplary references that are incorporated byreference: Waterman, Introduction to Computational Biology: Maps,Sequences and Genomes (Chapman & Hall/CRC, 1995); Pevzner, ComputationalMolecular Biology: An Algorithmic Approach (MIT Press, 2000); Drmanac,R., Labat, I., Crkvenjakov, R., J. Biomol. Struct. Dyn., 5: 1085,(1991); Reinert et al, J. Comput. Biol., 7: 1-46 (2000); Indury et al,J. Comput. Biol., 2: 291-306 (1995); Port et al, Genomics, 26: 84-100(1995); Waterman, Bull. Math. Biol., 56: 743-767 (1994); Vingron et al,J. Mol. Biol., 235: 1-12 (1994); Churchill et al, Genomics, 14: 89-98(1992); Lander et al, Genomics, 2: 231-239 (1988); Fleischmann et al,Science, 269: 496-512 (1995); and the like. In accordance with themethod of the invention, ordering sequence information obtained fromfinal fragments depends on the identity of at least one mixture of atleast one tier from which a final fragment is derived that gives rise toa portion of such sequence information. In one aspect, ordering suchinformation depends on a plurality of identifications of tiers andmixtures; and in another aspect, ordering such information depends onidentifying each mixture and each tier from which a final fragment isderived that provides sequence information.

In another aspect, as mentioned above, two tiers or levels are createdto assist in ordering sequence information in one or more targetpolynucleotides, which is implemented with the following steps: (a)fragmenting the one or more target polynucleotides present in apredetermined coverage amount to form a population containingoverlapping first-sized fragments each having an average lengthsubstantially less than those of the target polynucleotides; (b) forminga number of separate mixtures from the population of first-sizedfragments, such number being selected such that substantially everyfirst-sized fragment in a separate mixture is non-overlapping with everyother first-sized fragment of the same mixture; (c) fragmenting each ofthe first-sized fragments in each of the mixtures to form a populationof second-sized fragments for each mixture such that each second-sizedfragment has an average length substantially less than those of thefirst-sized fragments and such that the mixture of origin of each suchsecond-sized fragment can be identified; (d) determining sequenceinformation from at least a portion of one or more second-sizedfragments of each mixture; and (e) providing complete or partialnucleotide sequences of the one or more target polynucleotides byordering the sequence information from the aliquots, wherein suchordering depends on the mixture of origin of at least a portion of thesequence information. In one aspect, steps of fragmenting may include astep of separating the resulting fragments by size for more efficientfurther processing or so that subsequent aliquots contain differentaverage sized fragments, which can provide further useful orderinginformation, for example, if larger fragments are end-sequenced toprovide sequence segments with a known separation in the one or moretarget polynucleotides.

In one aspect, the methods of the invention provide physical maps of allDNA present in the sample. If the sample has one bacterial species, e.g.one bacterial chromosome then the method provides long range map forthat chromosome. If the sample has two bacterial strains, the methodprovides a map for both of them. Similarly the method provides a map ofboth parental sets of chromosomes in diploid genomes. Because genomicsequences usually have many repeats (including duplicated genes or exonsor other functional elements) physical mapping with high resolution andlong range is critical for complete unambiguous sequence assembly. Forgenomes with larger numbers of dispersed repeats longer than thesequence read length fragment lengths can adjusted so that there aremore fragments or more levels of fragmenting. By being able to assembleseparately parental chromosomes in diploid genomes, haplotypeinformation is obtained. In one aspect, the more similar the parentalgenomes are the longer initial fragment size is required to be able toassemble them separately. For example if the difference between parentalchromosomes is 1 in 100,000 bases, then fragments close to 1 mb in sizewould be used.

In another aspect of fragmentation, instead of having a single optimalDNA fragment length, a more informative approach may consist of having,for example, 10× coverage in, for example, 100-200 kb and 10× coveragein 30-50 kb fragments. For maximal informativeness each fragmentpreparation may be processed separately in their own set of tubes orwell-plates. Another approach is to start with, for example, 20× of, forexample, 200 kb long fragments distributed in say 96-wells. This assuresindependence of 40 fragments (20 from each parental chromosome) thatoverlap a given 200 kb fragment. For human genomes, each well will haveabout 6×20 Gb/200 kb/96 wells=˜10,000 200 kb fragments covering 20% ofeach parental genomes. These fragments can be further fragmented to10-30 kb fragments and then further aliquoted, for example, eachoriginal well into 4 new wells. This fragmenting and aliquoting stepprovides high frequency DNA brakes and reduces complexity of DNA perwell, which are both needed for efficient mapping of frequent repeats,but preserves 200 kb information (it is known which 4 final wells map toeach initial well) needed for assembly of parental chromosomes orsimilar bacterial genomes present in an environmental sample. Thisrepresents a three level fragmenting process.

In another aspect, the following is an example of using hierarchical DNAfragmentation and standard shotgun sequencing of clones. First-sizedfragments are subjected to “whole DNA” amplification method such as RCAto provide amplification in the range of 1000-100,000 fold. Theresulting DNA may be shorter than first-sized fragments. The resultingDNA is subjected to second fragmentation step using nucleases such asDNAse I or mechanical fragmentation to generate second-sized fragmentsin the range of 30-3000 bases. Second-sized fragments are “cloned”, e.g.ligated into a plasmid vector. Each aliquot uses a vector that hasdifferent tag sequence, or an adapter with such sequence is firstligated to second-sized fragments. The resulting DNA is mixed from someor all wells and transformed into host cells, resulting into individualcolonies harboring one cloned second-sized fragment. A set of coloniesthat provides at least about one fold coverage (and preferably 3-6 foldcoverage) of the first-sized fragments is collected, prepared forsequencing, and sequenced using dideoxy sequencing technology. Thesequencing primer(s) and reaction are adjusted to successfully read tagsequence in addition to at least one portion of the second-sizedfragment. The resulting sequences are grouped by the tag and sequencecontigs assembled in de-novo sequencing or are used to define areference sequence for first-sized fragments in DNA re-sequencingapplications. Overlapped sequence contigs from independent aliquots areused to assemble longer contigs to generate complete sequence orscaffolds of the genomic DNA present in the sample.

In one aspect, the methods of the invention use short and mediumsequence read length of massively parallel sequencing technologies toconstruct maps or orderings of such short reads in targetpolynucleotides. In another aspect, preparing fragments in accordancewith the invention in essence allows one to convert a short read lengthsequencing technique into long read length technique. That is, byaliquoting fragments so that the resulting fragments can be fullyreconstruct from the short read lengths available, the reconstructfragments can be used, in turn, to reconstruct fragments from the nexthigher level in a hierarchy.

For genomic sequencing using short read length techniques, it isimportant to incorporate effective mapping strategies to locate theshort reads to the full genome of the organism. This is especiallyimportant for analysis of complex diploid genomes, de novo sequencing,and sequencing mixtures of bacterial genomes. The hierarchicalfragmentation procedure addresses this issue. It may also aid inpredicting protein alleles and to map short reads to the correctpositions within the genome. Another example is the correct assignmentof a mutation in a gene family if it occurs within ˜100b DNA sequenceshared between multiple genes. This method represents an enablingtechnology that provides mapping information for assembling chromosomalhaplotypes for any sequencing method based on random DNA fragmentation.In this method, genomic DNA is first isolated as 30-300 kb sizedfragments. Through proper dilution, a small subset of these fragmentsare, at random, placed in discrete wells or multi-well plates or similaraccessories. For example a plate with 96, 384 or 1536 wells can be usedfor these fragment subsets. An optimal way to create these DNA aliquotsis to isolate the DNA with a method that naturally fragments to highmolecular weight forms, dilute to 10-30 genome equivalents afterquantitation, and then split the entire preparation into 384 wells. Thisshould result in a satisfactory representation of all genomic sequences,but performing DNA isolation on 10-30 cells with 100% recoveryefficiency would assure that all chromosomal regions are representedwith the same coverage. A goal of aliquoting is to minimize cases whereany two overlapping fragments from the same region of a chromosome areplaced in the same plate well. For diploid genomes represented with 10×coverage, there are 20 overlapping fragments on average to separate indistinct wells. If this sample was then distributed over a 384 wellplate, then each well would contain, on average, 1,562 fragments. Byforming 384 fractions in a standard 384-well plate there may only beabout a 1/400 chance that two overlapping fragments may end up in thesame well. Even if some matching fragments are placed in the same well,the other overlapping fragments from each chromosomal region may providethe necessary unique mapping information. The prepared groups of longfragments may be further cut to the final fragment size of about 300 to600 bases. To obtain 10× coverage of each fragment in a group, the DNAin each well may be amplified before final cutting using well-developedwhole genome amplification methods. All short fragments from one wellmay then be arrayed (as described below) and sequenced on one separateunit array or in one section of a larger continuous matrix. A compositearray of 384 unit arrays is ideal for parallel analysis of these groupsof fragments. In the assembly of long sequences representing parentalchromosomes, the algorithm may use the critical information that shortfragments detected in one unit array belong to a limited number oflonger continuous segments each representing a discreet portion of onechromosome. In almost all cases the homologous chromosomal segments maybe analyzed on different unit arrays. Long (˜100 kb) continuous initialsegments form a tailing pattern and provide sufficient mappinginformation to assemble each parental chromosome separately as depictedbelow by relying on about 100 polymorphic sites per 100 kb of DNA. Inthe following example dots represent 100-1000 consecutive bases that areidentical in corresponding segments.

Well 3 ......T........C..........C...G..........A......... Well 20  ....C........T..........T...A......   .G.........C... Well 157                   .......T...A......  ..G...   ...C........A...C...Well 258            ...C..........C...G..........A.........T........G...T...Wells 3 and 258 assemble chromosome 1 of Parent 1:

...T........C..........C...G..........A..........T........G...TWells 20 and 157 assemble chromosome 1 of Parent 2:

...C........T......  T...A..........G...    ...C........A...C...Random arrays (described below) prepared by two-level DNA fragmentingcombine the advantages of both BAC sequencing and shotgun sequencing ina simple and efficient way.

The DNA may be isolated from cells by standard procedures such as theQiagen Genomic-tip kit, or any other procedure that maintains intactfragments of approximately 30-300 kilobases in length. Pulse field gelelectrophoresis may be used to demonstrate effective size distributionof DNA fragments of several sample preparation methods. Genomic derivedDNA fragments of approximately 30-300 Kb in size and could be diluted to10-20 genome equivalents of DNA in an aliquot. The DNA may bedistributed over the wells of a 384 well plate. To confirm that thedistribution process has worked, PCR may be used to demonstrate that onaverage, 10 positive wells are identified per genomic region. However,since there is only 1 target per well this may be below the level ofsensitivity for many PCR assays, so random whole genome amplificationmethods may be applied to increase the initial copy number for PCRamplification.

Amplification of the single targets obtained in the chromosomalseparation procedure may be required for subsequent procedures. Toachieve this one may utilize existing, commercially available methodsfor whole genome amplification, preferably linear amplification methodsmay be used that produce 10-100 fold amplification. In effect, theseprocedures should not discriminate in terms of the sequences that are tobe amplified but instead may amplify all sequences within the sample.Note that this procedure does not require intact amplification of entire100 kb fragments. Amplification in the form of fragments as short as 1kb is sufficient.

Although each well contains amplified DNA, the sequence complexity ofeach well hasn't changed from the original 150-300 megabases. The nextphase is to further fragment amplified DNA into smaller fragments ofapproximately 300-600 bases in length. This may be achieved by Dnaseldigestion or possibly sonication. The DNA samples may then be heated togenerate single stranded DNA that is then ligated to the left and rightadapter subunits while in the single stranded state. The DNA within eachwell of the 384 well plate may be precipitated by isopropanol toselectively precipitate the longer fragments while the shorter fragmentsand adapters remain in the solution phase. The re-suspended DNA can thenbe attached to streptavidin coated magnetic beads for subsequentenzymatic procedures.

In another aspect, for simplified preparation of 384 genome fractions isto prepare 384 initial adapters encoded with a different 5-mer or 6-mersequence and use them in each of 384 wells. After the initial encodedadapter is ligated, DNA from all wells is mixed for further processingin a single tube including RCR. Instead of having 384 subarrayscorresponding to 384-wells the same information would be extracted bysequencing 5-6 bases using an additional adapter adjacent to these 5-6base.

In one aspect, methods of the invention are particularly useful in theassembly of target polynucleotides that contain repetitive sequenceregions, or repeat sequences, whose lengths are greater that of the readlength of the sequence analysis method being used, as illustrated in thefollowing example. At the same time, having DNA fragments starting onaverage every 5 kb provide a high resolution physical map for accuratesequence assembly in the presence of dispersed repeats longer than thesequence read length. Consider this situation with 100 kb fragmentscovering a genomic region with 4 identical 500 base repeats:

M1 ---70kb----500b repeat----4kb----500b repeat----6kb----500b-----20kb--- M2 ---10kb----500b repeat----4kb----500brepeat ----6kb----500b-----30kb ----500b repeat ---49kb---- M3         ----3kb----500b repeat ----6kb----500b-----30kb ----500b repeat---60kb---- M4                     ---2kb----500b-----30kb ----500brepeat ---68kb----If the read length is less than 500 bases, without mapping informationprovided by 100 kb fragments we will not be able to assemble this partof the sequence. In the case of resequencing, if one of the repeats hasa mutation we would not be able to tell which of the repeats is mutated.In the case of de-novo genome sequencing, we would not be able todetermine the order of the sequence segments starting and ending withthe repeat sequence. But having frequent beginnings and ends of longoverlapped fragments that are sequenced independently the completesequence information is obtained. In the above example, Fragment 1 endsbefore repeat 4, e.g. provides information which repeat is the lastrepeat in fragments 2-4. Fragment 3 tells which repeat is first infragments 1 and 2. Finally, fragment 4 maps the order of repeat 2 and 3in fragments 2 and 3. If fragment 4 started 2-3 kb downstream (e.g. ifwe do not have high coverage) we would not be able to map repeats 2 and3.

For assembly of parental chromosomes we need long overlaps and formapping repeats we need frequent brakes. There is an optimum of lengthfor given genome coverage. For 20× coverage 50 kb fragments provide 4times more brakes than 200 kb fragments (e.g. 1.25 kb instead of 5 kbaverage length of informational fragments; informational fragments aresequence segments between neighboring beginnings or ends of overlappingphysical genomics fragments; the average length of informationalfragments is calculated by the following equation: fragment length/2×coverage, e.g. 200 kb/2×20=5 kb) and such 50 kb fragments at 20×coverage may have long enough overlaps (47.5 kb, on average; almost allconsecutive overlaps >5 kb). Random fragmentation is simpler to get suchfrequent cutting of genomic DNA than to use 20 or more rare-cutterrestriction enzymes.

For viral analysis, when the entire genome is 10-100 kb, an importantgoal is determining the actual sequence of strains, e.g. an emergingstrain. Because there is no (or almost no) long repeats in short genomesfragmenting and overlaps many not be required. For bacterial genomeslong overlaps (30-100 kb) are needed, if it is desired to assemblecompletely individual genomes of highly similar strains (one sequencedifference at 1-10 kb). If the differences are more frequent, straingenomes can be assembled using shorter fragments, e.g. 10-30 kb, withshorter overlaps.

In one aspect of analyzing mixtures of bacterial genomes, aliquots maybe made so that each aliquot contains one cell type, or a maximum of onecell. DNA can then be fragmented to ˜100 kb in level 2, if thatinformation is needed for the sequence assembly across repeats. Such analiquoting strategy can be used, for example, for more accurate pathogendiagnostic for health or biodefense applications. Most of the time,pathogens (e.g. HIV or other virus) are present in mixtures, whencomplete viral or bacterial strain or haplotype determination iscritical to spot an emerging resistant organism or man-modified organismmixed with non-virulent natural strains.

In another aspect of the invention, a method is provided for preparingfor sequence analysis one or more target polynucleotides present in apredetermined coverage amount, such method comprising the followingsteps: (i) fragmenting the one or more target polynucleotides to form apopulation containing overlapping first-sized fragments each having anaverage length substantially less than those of the targetpolynucleotides; (ii) aliquoting the population of first-sized fragmentsinto a number of separate mixtures, such number being selected such thatsubstantially every first-sized fragment in a separate mixture isnon-overlapping with every other first-sized fragment of the sameseparate mixture; and (iii) attaching an oligonucleotide tag to eachfirst-sized fragment in each separate mixture so that theoligonucleotide tag identifies the separate mixture of the first-sizedfragment.

Short Read Length Sequencing Technique Using Random Arrays

In one aspect, the present invention may be used with “short readlength” sequencing techniques for analysis of long targetpolynucleotides. Of particular interest is a sequence analysis techniquedescribed below that makes use of a random array of DNA fragmentsderived from one or more target polynucleotides. It comprises theformation of one or more random arrays of single molecules that areconcatemer of DNA fragments derived from one or more targetpolynucleotides. Such concatemers are disposed randomly on a surface ofa support material, usually from a solution; thus, in one aspect, suchconcatemers are uniformly distributed on a surface in closeapproximation to a Poisson distribution. In another aspect, suchconcatemers are disposed on a surface that contains discrete spacedapart regions in which single molecules are attached. Preferably,concatemer sizes and compositions, preparation methods, and areas ofsuch discrete spaced apart regions are selected so that substantiallyall such regions contain at most only one single molecule. Concatemersof DNA fragments are roughly in a random coil configuration on a surfaceand are confined to the area of a discrete spaced apart region. In oneaspect, the discrete space apart regions have defined locations in aregular array, which may correspond to a rectilinear pattern, hexagonalpattern, or the like. A regular array of such regions is advantageousfor detection and data analysis of signals collected from the arraysduring an analysis. Also, concatemers confined to the restricted area ofa discrete spaced apart region provide a more concentrated or intensesignal, particularly when fluorescent probes are used in analyticaloperations, thereby providing higher signal-to-noise values. Concatemersare randomly distributed on the discrete spaced apart regions so that agiven region usually is equally likely to receive any of the differentsingle molecules. In other words, the resulting arrays are not spatiallyaddressable immediately upon fabrication, but may be made so by carryingout an identification or decoding operation. That is, the identities ofthe concatemers are discernable, but not known. Concatemers have sizesmay range from a few thousand nucleotides, e.g. 10,000, to severalhundred thousand nucleotides, e.g. 100-200 thousand.

The above concepts are illustrated more fully in the embodiments shownschematically in FIGS. 3A-3C. After describing these figures, elementsof the invention are disclosed in additional detail and examples aregiven. As mentioned above, in one aspect, macromolecular structures ofthe invention are single stranded polynucleotides comprising concatemersof a target sequence or fragment. In particular, such polynucleotidesmay be concatemers of a target sequence and an adaptor oligonucleotide.For example, source nucleic acid (1000) is treated (1001) to form singlestranded fragments (1006), preferably in the range of from 50 to 600nucleotides, and more preferably in the range of from 300 to 600nucleotides, which are then ligated to adaptor oligonucleotides (1004)to form a population of adaptor-fragment conjugates (1002). Sourcenucleic acid (1000) may be genomic DNA extracted from a sample usingconventional techniques, or a cDNA or genomic library produced byconventional techniques, or synthetic DNA, or the like. Treatment (1001)usually entails fragmentation by a conventional technique, such aschemical fragmentation, enzymatic fragmentation, or mechanicalfragmentation, followed by denaturation to produce single stranded DNAfragments. Adaptor oligonucleotides (1004), in this example, are used toform (1008) a population (1010) of DNA circles by the method illustratedin FIG. 2A. In one aspect, each member of population (1010) has anadaptor with an identical primer binding site and a DNA fragment fromsource nucleic acid (1000). The adapter also may have other functionalelements including, but not limited to, tagging sequences, attachmentsequences, palindromic sequences, restriction sites, functionalizationsequences, and the like. In other embodiments, classes of DNA circlesmay be created by providing adaptors having different primer bindingsites. After DNA circles (1010) are formed, a primer and rolling circlereplication (RCR) reagents may be added to generate (1011) in aconventional RCR reaction a population (1012) of concatemers (1015) ofthe complements of the adaptor oligonucleotide and DNA fragments, whichpopulation can then be isolated using conventional separationtechniques. Alternatively, RCR may be implemented by successive ligationof short oligonucleotides, e.g. 6-mers, from a mixture containing allpossible sequences, or if circles are synthetic, a limited mixture ofoligonucleotides having selected sequences for circle replication.Concatemers may also be generated by ligation of target DNA in thepresence of a bridging template DNA complementary to both beginning andend of the target molecule. A population of different target DNA may beconverted in concatemers by a mixture of corresponding bridgingtemplates. Isolated concatemers (1014) are then disposed (1016) ontosupport surface (1018) to form a random array of single molecules.Attachment may also include wash steps of varying stringencies to removeincompletely attached single molecules or other reagents present fromearlier preparation steps whose presence is undesirable or that arenonspecifically bound to surface (1018). Concatemers (1020) can be fixedto surface (1018) by a variety of techniques, including covalentattachment and non-covalent attachment. In one embodiment, surface(1018) may have attached capture oligonucleotides that form complexes,e.g. double stranded duplexes, with a segment of the adaptoroligonucleotide, such as the primer binding site or other elements. Inother embodiments, capture oligonucleotides may comprise oligonucleotideclamps, or like structures, that form triplexes with adaptoroligonucleotides, e.g. Gryaznov et al, U.S. Pat. No. 5,473,060. Inanother embodiment, surface (1018) may have reactive functionalitiesthat react with complementary functionalities on the concatemers to forma covalent linkage, e.g. by way of the same techniques used to attachcDNAs to microarrays, e.g. Smirnov et al (2004), Genes, Chromosomes &Cancer, 40: 72-77; Beaucage (2001), Current Medicinal Chemistry, 8:1213-1244, which are incorporated herein by reference. Long DNAmolecules, e.g. several hundred nucleotides or larger, may also beefficiently attached to hydrophobic surfaces, such as a clean glasssurface that has a low concentration of various reactivefunctionalities, such as —OH groups. Concatemers of DNA fragments may befurther amplified in situ after disposition of a surface. For exampleafter disposition, concatemer may be cleaved by reconstituting arestriction site in adaptor sequences by hybridization of anoligonucleotide, after which the fragments are circularized as describedbelow and amplified in situ by a RCR reaction.

FIG. 3B illustrates a section (1102) of a surface of a random array ofsingle molecules, such as single stranded polynucleotides. Suchmolecules under conventional conditions (a conventional DNA buffer, e.g.TE, SSC, SSPE, or the like, at room temperature) form random coils thatroughly fill a spherical volume in solution having a diameter of fromabout 100 to 300 nm, which depends on the size of the DNA and bufferconditions, in a manner well known in the art, e.g. Edvinsson, “On thesize and shape of polymers and polymer complexes,” Dissertation 696(University of Uppsala, 2002). One measure of the size of a random coilpolymer, such as single stranded DNA, is a root mean square of theend-to-end distance, which is roughly a measure of the diameter of therandomly coiled structure. Such diameter, referred to herein as a“random coil diameter,” can be measured by light scatter, usinginstruments, such as a Zetasizer Nano System (Malvem Instruments, UK),or like instrument. Additional size measures of macromolecularstructures of the invention include molecular weight, e.g. in Daltons,and total polymer length, which in the case of a branched polymer is thesum of the lengths of all its branches. Upon attachment to a surface,depending on the attachment chemistry, density of linkages, the natureof the surface, and the like, single stranded polynucleotides fill aflattened spheroidal volume that on average is bounded by a region(1107) defined by dashed circles (1108) having a diameter (1110), whichis approximately equivalent to the diameter of a concatemer in randomcoil configuration. Stated another way, in one aspect, macromolecularstructures, e.g. concatemers, and the like, are attached to surface(1102) within a region that is substantially equivalent to a projectionof its random coil state onto surface (1102), for example, asillustrated by dashed circles (1108). An area occupied by amacromolecular structure can vary, so that in some embodiments, anexpected area may be within the range of from 2-3 times the area ofprojection (1108) to some fraction of such area, e.g. 25-50 percent. Asmentioned else where, preserving the compact form of the macromolecularstructure on the surface allows a more intense signal to be produced byprobes, e.g. fluorescently labeled oligonucleotides, specificallydirected to components of a macromolecular structure or concatemer. Thesize of diameter (1110) of regions (1107) and distance (1106) to thenearest neighbor region containing a single molecule are two quantitiesof interest in the fabrication of arrays. A variety of distance metricsmay be employed for measuring the closeness of single molecules on asurface, including center-to-center distance of regions (1107),edge-to-edge distance of regions (1007), and the like. Usually,center-to-center distances are employed herein. The selection of theseparameters in fabricating arrays of the invention depends in part on thesignal generation and detection systems used in the analyticalprocesses. Generally, densities of single molecules are selected thatpermit at least twenty percent, or at least thirty percent, or at leastforty percent, or at least a majority of the molecules to be resolvedindividually by the signal generation and detection systems used. In oneaspect, a density is selected that permits at least seventy percent ofthe single molecules to be individually resolved. In one aspect,whenever scanning electron microscopy is employed, for example, withmolecule-specific probes having gold nanoparticle labels, e.g. Nie et al(2006), Anal. Chem., 78: 1528-1534, which is incorporated by reference,a density is selected such that at least a majority of single moleculeshave a nearest neighbor distance of 50 nm or greater; and in anotheraspect, such density is selected to ensure that at least seventy percentof single molecules have a nearest neighbor distance of 100 nm orgreater. In another aspect, whenever optical microscopy is employed, forexample with molecule-specific probes having fluorescent labels, adensity is selected such that at least a majority of single moleculeshave a nearest neighbor distance of 200 nm or greater; and in anotheraspect, such density is selected to ensure that at least seventy percentof single molecules have a nearest neighbor distance of 200 nm orgreater. In still another aspect, whenever optical microscopy isemployed, for example with molecule-specific probes having fluorescentlabels, a density is selected such that at least a majority of singlemolecules have a nearest neighbor distance of 300 nm or greater; and inanother aspect, such density is selected to ensure that at least seventypercent of single molecules have a nearest neighbor distance of 300 nmor greater, or 400 nm or greater, or 500 nm or greater, or 600 nm orgreater, or 700 nm or greater, or 800 nm or greater. In still anotherembodiment, whenever optical microscopy is used, a density is selectedsuch that at least a majority of single molecules have a nearestneighbor distance of at least twice the minimal feature resolution powerof the microscope. In another aspect, polymer molecules of the inventionare disposed on a surface so that the density of separately detectablepolymer molecules is at least 1000 per μm², or at least 10,000 per μm²,or at least 100,000 per μm².

In another aspect of the invention, illustrated for a particularembodiment in FIG. 3C, the requirement of selecting densities ofrandomly disposed single molecules to ensure desired nearest neighbordistances is obviated by providing on a surface discrete spaced apartregions that are substantially the sole sites for attaching singlemolecules. That is, in such embodiments the regions on the surfacebetween the discrete spaced apart regions, referred to herein as“inter-regional areas,” are inert in the sense that concatemers, orother macromolecular structures, do not bind to such regions. In someembodiments, such inter-regional areas may be treated with blockingagents, e.g. DNAs unrelated to concatemer DNA, other polymers, and thelike As in FIG. 1A, source nucleic acids (1000) are fragmented andadaptored (1002) for circularization (1010), after which concatemers areformed by RCR (1012). Isolated concatemers (1014) are then applied tosurface (1120) that has a regular array of discrete spaced apart regions(1122) that each have a nearest neighbor distance (1124) that isdetermined by the design and fabrication of surface (1120). As describedmore fully below, arrays of discrete spaced apart regions (1122) havingmicron and submicron dimensions for derivatizing with captureoligonucleotides or reactive functionalities can be fabricated usingconventional semiconductor fabrication techniques, including electronbeam lithography, nano imprint technology, photolithography, and thelike. Generally, the area of discrete spaced apart regions (1122) isselected, along with attachment chemistries, macromolecular structuresemployed, and the like, to correspond to the size of single molecules ofthe invention so that when single molecules are applied to surface(1120) substantially every region (1122) is occupied by no more than onesingle molecule. The likelihood of having only one single molecule perdiscrete spaced apart region may be increased by selecting a density ofreactive functionalities or capture oligonucleotides that results infewer such moieties than their respective complements on singlemolecules. Thus, a single molecule will “occupy” all linkages to thesurface at a particular discrete spaced apart region, thereby reducingthe chance that a second single molecule will also bind to the sameregion. In particular, in one embodiment, substantially all the captureoligonucleotides in a discrete spaced apart region hybridize to adaptoroligonucleotides a single macromolecular structure. In one aspect, adiscrete spaced apart region contains a number of reactivefunctionalities or capture oligonucleotides that is from about tenpercent to about fifty percent of the number of complementaryfunctionalities or adaptor oligonucleotides of a single molecule. Thelength and sequence(s) of capture oligonucleotides may vary widely, andmay be selected in accordance with well known principles, e.g. Wetmur,Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259(1991); Britten and Davidson, chapter 1 in Hames et al, editors, NucleicAcid Hybridization: A Practical Approach (IRL Press, Oxford, 1985). Inone aspect, the lengths of capture oligonucleotides are in a range offrom 6 to 30 nucleotides, and in another aspect, within a range of from8 to 30 nucleotides, or from 10 to 24 nucleotides. Lengths and sequencesof capture oligonucleotides are selected (i) to provide effectivebinding of macromolecular structures to a surface, so that losses ofmacromolecular structures are minimized during steps of analyticaloperations, such as washing, etc., and (ii) to avoid interference withanalytical operations on analyte molecules, particularly when analytemolecules are DNA fragments in a concatemer. In regard to (i), in oneaspect, sequences and lengths are selected to provide duplexes betweencapture oligonucleotides and their complements that are sufficientlystable so that they do not dissociate in a stringent wash. In regard to(ii), if DNA fragments are from a particular species of organism, thendatabases, when available, may be used to screen potential capturesequences that may form spurious or undesired hybrids with DNAfragments. Other factors in selecting sequences for captureoligonucleotides are similar to those considered in selecting primers,hybridization probes, oligonucleotide tags, and the like, for whichthere is ample guidance, as evidenced by the references cited below inthe Definitions section.

In one aspect, the area of discrete spaced apart regions (1122) is lessthan 1 μm²; and in another aspect, the area of discrete spaced apartregions (1122) is in the range of from 0.04 μm² to 1 μm²; and in stillanother aspect, the area of discrete spaced apart regions (1122) is inthe range of from 0.2 μm² to 1 μm². In another aspect, when discretespaced apart regions are approximately circular or square in shape sothat their sizes can be indicated by a single linear dimension, the sizeof such regions are in the range of from 125 nm to 250 nm, or in therange of from 200 nm to 500 nm. In one aspect, center-to-centerdistances of nearest neighbors of regions (1122) are in the range offrom 0.25 μm to 20 μm; and in another aspect, such distances are in therange of from 1 μm to 10 μm, or in the range from 50 to 1000 nm. In oneaspect, regions (1120) may be arranged on surface (1018) in virtuallyany pattern in which regions (1122) have defined locations, i.e. in anyregular array, which makes signal collection and data analysis functionsmore efficient. Such patterns include, but are not limited to,concentric circles of regions (1122), spiral patterns, rectilinearpatterns, hexagonal patterns, and the like. Preferably, regions (1122)are arranged in a rectilinear or hexagonal pattern.

Source Nucleic Acids and Circularization of Target Sequences

Target polynucleotides for analysis may be extracted or derived from asample, such as genomic DNA or cDNAs, from a patient, from anenvironmental sample, or from an organism of economic interest, or thelike. As diagrammed in FIG. 1C, random arrays comprising concatemers ofDNA fragments from such samples are useful in providing genome-wideanalyses, including sequence determination, SNP measurement, allelequantitation, copy number measurements, and the like. Formammalian-sized genomes (150), fragmentation is carried out in at leasttwo stages, a first stage to generate a population (152) of fragments ina size range of from about 100 kilobases (Kb) to about 250 kilobases,and a second stage after aliquoting (154), applied separately to the100-250 Kb fragments, to generate fragments (156) in the size range offrom about 50 to 600 nucleotides, and more preferably in the range offrom about 300 to 600 nucleotides, which are then used to generateconcatemers for a random array. The amount of genomic DNA required forconstructing arrays of the invention can vary widely. In one aspect, formammalian-sized genomes, fragments are generated from at least 10genome-equivalents of DNA; and in another aspect, fragments aregenerated from at least 30 genome-equivalents of DNA; and in anotheraspect, fragments are generated from at least 60 genome-equivalents ofDNA (i.e. 10, 30, or 60 coverage amounts).

For mammalian-sized genomes, an initial fragmentation of genomic DNA canbe achieved by digestion with one or more “rare” cutting restrictionendonucleases, such as Not I, Asc I, Bae I, CspC I, Pac I, Fse I, Sap I,Sfi I, Psr I, or the like, to produce fragments that are targetpolynucleotides for analysis by the invention; that is, such restrictionfragments are processed by the random fragmentation techniques describedabove. Specific fragments may be isolated from such digested DNA forsubsequent processing as illustrated in FIG. 2B. Genomic DNA (230) isdigested (232) with a rare cutting restriction endonuclease to generatefragments (234), after which the fragments (234) are further digestedfor a short period (i.e. the reaction is not allowed to run tocompletion) with a 5′ single stranded exonuclease, such as Xexonuclease, to expose sequences (237) adjacent to restriction sitesequences at the end of the fragments. Such exposed sequences will beunique for each fragment. Accordingly, biotinylated primers (241)specific for the ends of desired fragments can be annealed to a captureoligonucleotide for isolation; or alternatively, such fragments can beannealed to a primer having a capture moiety, such as biotin, andextended with a DNA polymerase that does not have strand displacementactivity, such as Taq polymerase Stoffel fragment. After such extension,the 3′ end of primers (241) abut the top strand of fragments (242) suchthat they can be ligated to form a continuous strand. The latterapproach may also be implemented with a DNA polymerase that does havestrand displacement activity and replaces the top strand (242) bysynthesis. In either approach, the biotinylated fragments may then beisolated (240) using a solid support (239) derivatized withstreptavidin.

In another aspect, primer extension from a genomic DNA template is usedto generate a linear amplification of selected sequences greater than 10kilobases surrounding genomic regions of interest. For example, tocreate a population of defined-sized targets, 20 cycles of linearamplification is performed with a forward primer followed by 20 cycleswith a reverse primer. Before applying the second primer, the firstprimer is removed with a standard column for long DNA purification ordegraded if a few uracil bases are incorporated. A greater number ofreverse strands are generated relative to forward strands resulting in apopulation of double stranded molecules and single stranded reversestrands. The reverse primer may be biotinylated for capture tostreptavidin beads which can be heated to melt any double strandedhomoduplexes from being captured. All attached molecules will be singlestranded and representing one strand of the original genomic DNA.

The products produced can be fragmented to 0.2-2 kb in size, or morepreferably, 0.3-0.6 kb in size (effectively releasing them from thesolid support) and circularized for an RCR reaction. In one method ofcircularization, illustrated in FIG. 2A, after genomic DNA (200) isfragmented and denatured (202), single stranded DNA fragments (204) arefirst treated with a terminal transferase (206) to attach a poly dAtails (208) to 3-prime ends. This is then followed by ligation (212) ofthe free ends intra-molecularly with the aid of bridging oligonucleotide(210). that is complementary to the poly dA tail at one end andcomplementary to any sequence at the other end by virtue of a segment ofdegenerate nucleotides. Duplex region (214) of bridging oligonucleotide(210) contains at least a primer binding site for RCR and, in someembodiments, sequences that provide complements to a captureoligonucleotide, which may be the same or different from the primerbinding site sequence, or which may overlap the primer binding sitesequence. The length of capture oligonucleotides may vary widely, In oneaspect, capture oligonucleotides and their complements in a bridgingoligonucleotide have lengths in the range of from 10 to 100 nucleotides;and more preferably, in the range of from 10 to 40 nucleotides. In someembodiments, duplex region (214) may contain additional elements, suchas an oligonucleotide tag, for example, for identifying the sourcenucleic acid from which its associated DNA fragment came. That is, insome embodiments, circles or adaptor ligation or concatemers fromdifferent source nucleic acids may be prepared separately during which abridging adaptor containing a unique tag is used, after which they aremixed for concatemer preparation or application to a surface to producea random array. The associated fragments may be identified on such arandom array by hybridizing a labeled tag complement to itscorresponding tag sequences in the concatemers, or by sequencing theentire adaptor or the tag region of the adaptor. Circular products (218)may be conveniently isolated by a conventional purification column,digestion of non-circular DNA by one or more appropriate exonucleases,or both.

As mentioned above, DNA fragments of the desired sized range, e.g.50-600 nucleotides, can also be circularized using circularizingenzymes, such as CircLigase, as single stranded DNA ligase thatcircularizes single stranded DNA without the need of a template.CircLigase is used in accordance with the manufacterer's instructions(Epicentre, Madison, Wis.). A preferred protocol for forming singlestranded DNA circles comprising a DNA fragment and one or more adaptersis to use standard ligase such as T4 ligase for ligation an adapter toone end of DNA fragment and than to use CircLigase to close the circle,as described more fully below.

An exemplary protocol for generating a DNA circle comprising an adaptoroligonucleotide and a target sequence using T4 ligase. The targetsequence is a synthetic oligo T1N (sequence:5′-NNNNNNNGCATANCACGANGTCATNATCGTNCAAACGTCAGTCCANGAATCNAGATCCACTTAGANTGNCGNNNNNNNN-3′ (SEQ ID NO: 1)). The adaptor is made up of 2separate oligos. The adaptor oligo that joins to the 5′ end of T1N isBR2-ad (sequence:5′-TATCATCTGGATGTTAGGAAGACAAAAGGAAGCTGAGGACATTAACGGAC-3′ (SEQ ID NO: 2))and the adaptor oligo that joins to the 3′ end of T1N is UR3-ext(sequence: 5′-ACCTTCAGACCAGAT-3′ (SEQ ID NO: 3)) UR3-ext contains a typeIIs restriction enzyme site (Acu I: CTTCAG) to provide a way tolinearize the DNA circular for insertion of a second adaptor. BR2-ad isannealed to BR2-temp (sequence 5′-NNNNNNNGTCCGTTAATGTCCTCAG-3′ (SEQ IDNO: 4)) to form a double-stranded adaptor BR2 adaptor. UR3-ext isannealed to biotinylated UR3-temp (sequence5′-[BIOTIN]ATCTGGTCTGAAGGTNNNNNN-3′ (SEQ ID NO: 5)) to form adouble-stranded adaptor UR3 adaptor. 1 μmol of target T1N is ligated to25 μmol of BR2 adaptor and 10 μmol of UR3 adaptor in a single ligationreaction containing 50 mM Tris-Cl, pH7.8, 10% PEG, 1 mM ATP, 50 mg/LBSA, 10 mM MgCl₂, 0.3 unit/μl T4 DNA ligase (Epicentre Biotechnologies,WI) and 10 mM DTT) in a final volume of 10 ul. The ligation reaction isincubated in a temperature cycling program of 15° C. for 11 min, 37° C.for 1 min repeated 18 times. The reaction is terminated by heating at70° C. for 10 min. Excess BR2 adaptors are removed by capturing theligated products with streptavidin magnetic beads (New England Biolabs,MA). 3.3 ul of 4× binding buffer (2M NaCl, 80 mM Tris HCl pH7.5) isadded to the ligation reaction which is then combined with 15 μg ofstreptavidin magnetic beads in 1× binding buffer (0.5M NaCl, 20 mM TrisHCl pH7.5). After 15 min incubation in room temperature, the beads arewashed twice with 4 volumes of low salt buffer (0.15M NaCl, 20 mM TrisHCl pH7.5). Elution buffer (10 mM Tris HCl pH7.5) is pre-warmed to 70deg, 10 μl of which is added to the beads at 70° C. for 5 min. Aftermagnetic separation, the supernatant is retained as primary purifiedsample. This sample is further purified by removing the excess UR3adaptors with magnetic beads pre-bound with a biotinylated oligoBR-rc-bio (sequence: 5′-[BIOTIN]CTTTTGTCTTCCTAACATCC-3′ (SEQ ID NO: 6))that is reverse complementary to BR2-ad similarly as described above.The concentration of the adaptor-target ligated product in the finalpurified sample is estimated by urea polyacrylamide gel electrophoresisanalysis. The circularization is carried out by phosphorylating theligation products using 0.2 unit/μl T4 polynucleotide kinase (EpicentreBiotechnologies) in 1 mM ATP and standard buffer provided by thesupplier, and circularized with ten-fold molar excess of a splint oligoUR3-closing-88 (sequence 5′-AGATGATAATCTGGTC-3′ (SEQ ID NO: 7)) using0.3 unit/μl of T4 DNA ligase (Epicentre Biotechnologies) and 1 mM ATP.The circularized product is validated by performing RCR reactions asdescribed below.

Guidance for selecting conditions and reagents for RCR reactions isavailable in many references available to those of ordinary skill, asevidence by the following that are incorporated by reference: Kool, U.S.Pat. No. 5,426,180; Lizardi, U.S. Pat. Nos. 5,854,033 and 6,143,495;Landegren, U.S. Pat. No. 5,871,921; and the like. Generally, RCRreaction components comprise single stranded DNA circles, one or moreprimers that anneal to DNA circles, a DNA polymerase having stranddisplacement activity to extend the 3′ ends of primers annealed to DNAcircles, nucleoside triphosphates, and a conventional polymerasereaction buffer. Such components are combined under conditions thatpermit primers to anneal to DNA circles and be extended by the DNApolymerase to form concatemers of DNA circle complements. An exemplaryRCR reaction protocol is as follows: In a 50 μL reaction mixture, thefollowing ingredients are assembled: 2-50 μmol circular DNA, 0.5units/μL phage φ29 DNA polymerase, 0.2 μg/μL BSA, 3 mM DNTP, 1×φ29 DNApolymerase reaction buffer (Amersham). The RCR reaction is carried outat 30° C. for 12 hours. In some embodiments, the concentration ofcircular DNA in the polymerase reaction may be selected to be low(approximately 10-100 billion circles per ml, or 10-100 circles perpicoliter) to avoid entanglement and other intermolecular interactions.

Preferably, concatemers produced by RCR are approximately uniform insize; accordingly, in some embodiments, methods of making arrays of theinvention may include a step of size-selecting concatemers. For example,in one aspect, concatemers are selected that as a population have acoefficient of variation in molecular weight of less than about 30%; andin another embodiment, less than about 20%. In one aspect, sizeuniformity is further improved by adding low concentrations of chainterminators, such ddNTPs, to the RCR reaction mixture to reduce thepresence of very large concatemers, e.g. produced by DNA circles thatare synthesized at a higher rate by polymerases. In one embodiment,concentrations of ddNTPs are used that result in an expected concatemersize in the range of from 50-250 Kb, or in the range of from 50-100 Kb.In another aspect, concatemers may be enriched for a particular sizerange using a conventional separation techniques, e.g. size-exclusionchromatography, membrane filtration, or the like.

Solid Phase Surfaces for Constructing Random Arrays

A wide variety of supports may be used with the invention. In oneaspect, supports are rigid solids that have a surface, preferably asubstantially planar surface so that single molecules to be interrogatedare in the same plane. The latter feature permits efficient signalcollection by detection optics, for example. In another aspect, solidsupports of the invention are nonporous, particularly when random arraysof single molecules are analyzed by hybridization reactions requiringsmall volumes. Suitable solid support materials include materials suchas glass, polyacrylamide-coated glass, ceramics, silica, silicon,quartz, various plastics, and the like. In one aspect, the area of aplanar surface may be in the range of from 0.5 to 4 cm². In one aspect,the solid support is glass or quartz, such as a microscope slide, havinga surface that is uniformly silanized. This may be accomplished usingconventional protocols, e.g. acid treatment followed by immersion in asolution of 3-glycidoxypropyl trimethoxysilane,N,N-diisopropylethylamine, and anhydrous xylene (8:1:24 v/v) at 80° C.,which forms an epoxysilanized surface. e.g. Beattie et a (1995),Molecular Biotechnology, 4: 213. Such a surface is readily treated topermit end-attachment of capture oligonucleotides, e.g. by providingcapture oligonucleotides with a 3′ or 5′ triethylene glycol phosphorylspacer (see Beattie et al, cited above) prior to application to thesurface. Many other protocols may be used for adding reactivefunctionalities to glass and other surfaces, as evidenced by thedisclosure in Beaucage (cited above).

Whenever enzymatic processing is not required, capture oligonucleotidesmay comprise non-natural nucleosidic units and/or linkages that conferfavorable properties, such as increased duplex stability; such compoundsinclude, but not limited to, peptide nucleic acids (PNAs), lockednucleic acids (LNA), oligonucleotide N3′→P5′ phosphoramidates,oligo-2′-O-alkylribonucleotides, and the like.

In embodiments of the invention in which patterns of discrete spacedapart regions are required, photolithography, electron beam lithography,nano imprint lithography, and nano printing may be used to generate suchpatterns on a wide variety of surfaces, e.g. Pirrung et al, U.S. Pat.No. 5,143,854; Fodor et al, U.S. Pat. No. 5,774,305; Guo, (2004) Journalof Physics D: Applied Physics, 37: R123-141; which are incorporatedherein by reference.

In one aspect, surfaces containing a plurality of discrete spaced apartregions are fabricated by photolithography. A commercially available,optically flat, quartz substrate is spin coated with a 100-500 nm thicklayer of photo-resist. The photo-resist is then baked on to the quartzsubstrate. An image of a reticle with a pattern of regions to beactivated is projected onto the surface of the photo-resist, using astepper. After exposure, the photo-resist is developed, removing theareas of the projected pattern which were exposed to the UV source. Thisis accomplished by plasma etching, a dry developing technique capable ofproducing very fine detail. The substrate is then baked to strengthenthe remaining photo-resist. After baking, the quartz wafer is ready forfunctionalization. The wafer is then subjected to vapor-deposition of3-aminopropyldimethylethoxysilane. The density of the aminofunctionalized monomer can be tightly controlled by varying theconcentration of the monomer and the time of exposure of the substrate.Only areas of quartz exposed by the plasma etching process may reactwith and capture the monomer. The substrate is then baked again to curethe monolayer of amino-functionalized monomer to the exposed quartz.After baking, the remaining photo-resist may be removed using acetone.Because of the difference in attachment chemistry between the resist andsilane, aminosilane-functionalized areas on the substrate may remainintact through the acetone rinse. These areas can be furtherfunctionalized by reacting them with p-phenylenediisothiocyanate in asolution of pyridine and N—N-dimethlyformamide. The substrate is thencapable of reacting with amine-modified oligonucleotides. Alternatively,oligonucleotides can be prepared with a 5′-carboxy-modifier-c10 linker(Glen Research). This technique allows the oligonucleotide to beattached directly to the amine modified support, thereby avoidingadditional functionalization steps.

In another aspect, surfaces containing a plurality of discrete spacedapart regions are fabricated by nano-imprint lithography (NIL). For DNAarray production, a quartz substrate is spin coated with a layer ofresist, commonly called the transfer layer. A second type of resist isthen applied over the transfer layer, commonly called the imprint layer.The master imprint tool then makes an impression on the imprint layer.The overall thickness of the imprint layer is then reduced by plasmaetching until the low areas of the imprint reach the transfer layer.Because the transfer layer is harder to remove than the imprint layer,it remains largely untouched. The imprint and transfer layers are thenhardened by heating. The substrate is then put into a plasma etcheruntil the low areas of the imprint reach the quartz. The substrate isthen derivatized by vapor deposition as described above.

In another aspect, surfaces containing a plurality of discrete spacedapart regions are fabricated by nano printing. This process uses photo,imprint, or e-beam lithography to create a master mold, which is anegative image of the features required on the print head. Print headsare usually made of a soft, flexible polymer such aspolydimethylsiloxane (PDMS). This material, or layers of materialshaving different properties, are spin coated onto a quartz substrate.The mold is then used to emboss the features onto the top layer ofresist material under controlled temperature and pressure conditions.The print head is then subjected to a plasma based etching process toimprove the aspect ratio of the print head, and eliminate distortion ofthe print head due to relaxation over time of the embossed material.Random array substrates are manufactured using nano-printing bydepositing a pattern of amine modified oligonucleotides onto ahomogenously derivatized surface. These oligo-nucleotides would serve ascapture probes for the RCR products. One potential advantage tonano-printing is the ability to print interleaved patterns of differentcapture probes onto the random array support. This would be accomplishedby successive printing with multiple print heads, each head having adiffering pattern, and all patterns fitting together to form the finalstructured support pattern. Such methods allow for some positionalencoding of DNA elements within the random array. For example, controlconcatemers containing a specific sequence can be bound at regularintervals throughout a random array.

Detection Instrumentation

As mentioned above, signals from single molecules on random arrays madein accordance with the invention are generated and detected by a numberof detection systems, including, but not limited to, scanning electronmicroscopy, near field scanning optical microscopy (NSOM), totalinternal reflection fluorescence microscopy (TIRFM), and the like.Abundant guidance is found in the literature for applying suchtechniques for analyzing and detecting nanoscale structures on surfaces,as evidenced by the following references that are incorporated byreference: Reimer et al, editors, Scanning Electron Microscopy: Physicsof Image Formation and Microanalysis, 2^(nd) Edition (Springer, 1998);Nie et al, Anal. Chem., 78: 1528-1534 (2006); Hecht et al, JournalChemical Physics, 112: 7761-7774 (2000); Zhu et al, editors, Near-FieldOptics: Principles and Applications (World Scientific Publishing,Singapore, 1999); Drmanac, International patent publication WO2004/076683; Lehr et al, Anal. Chem., 75: 2414-2420 (2003); Neuschaferet al, Biosensors & Bioelectronics, 18: 489-497 (2003); Neuschafer etal, U.S. Pat. No. 6,289,144; and the like. Of particular interest isTIRFM, for example, as disclosed by Neuschafer et al, U.S. Pat. No.6,289,144; Lehr et al (cited above); and Drmanac, International patentpublication WO 2004/076683. In one aspect, instruments for use witharrays of the invention comprise three basic components: (i) a fluidicssystem for storing and transferring detection and processing reagents,e.g. probes, wash solutions, and the like, to an array; (ii) a reactionchamber, or flow cell, holding or comprising an array and havingflow-through and temperature control capability; and (iii) anillumination and detection system. In one embodiment, a flow cell has atemperature control subsystem with ability to maintain temperature inthe range from about 5-95° C., or more specifically 10-85° C., and canchange temperature with a rate of about 0.5-2° C. per second. Anexemplary detection and imaging systems comprises a 100× objective usingTIRF or epi illumination and a 1.3 mega pixel Hamamatsu orca-er-ag on aZeiss axiovert 200, or like system.

Sequence Analysis of Random Arrays of Target Sequence Concatemers

As mentioned above, random arrays of biomolecules, such as genomic DNAfragments or cDNA fragments, provides a platform for large scalesequence determination and for genome-wide measurements based oncounting sequence tags, in a manner similar to measurements made byserial analysis of gene expression (SAGE) or massively parallelsignature sequencing, e.g. Velculescu, et al, (1995), Science 270,484-487; and Brenner et al (2000), Nature Biotechnology, 18: 630-634.Such genome-wide measurements include, but are not limited to,determination of polymorphisms, including nucleotide substitutions,deletions, and insertions, inversions, and the like, determination ofmethylation patterns, copy number patterns, and the like.

A variety of sequencing methodologies can be used with random arrays ofthe invention, including, but not limited to, hybridization-basedmethods, such as disclosed in Drmanac, U.S. Pat. Nos. 6,864,052;6,309,824; and 6,401,267; and Drmanac et al, U.S. patent publication2005/0191656, which are incorporated by reference, sequencing bysynthesis methods, e.g. Nyren et al, U.S. Pat. No. 6,210,891; Ronaghi,U.S. Pat. No. 6,828,100; Ronaghi et al (1998), Science, 281: 363-365;Balasubramanian, U.S. Pat. No. 6,833,246; Quake, U.S. Pat. No.6,911,345; Li et al, Proc. Natl. Acad. Sci., 100: 414-419 (2003), whichare incorporated by reference, and ligation-based methods, e.g. Shendureet al (2005), Science, 309: 1728-1739, which is incorporated byreference.

In one aspect, parallel sequencing of polynucleotide analytes ofconcatemers on a random array is accomplished by combinatorial SBH(cSBH), as disclosed by Drmanac in the above-cited patents. In oneaspect, a first and second sets of oligonucleotide probes are provide,wherein each sets has member probes that comprise oligonucleotideshaving every possible sequence for the defined length of probes in theset. For example, if a set contains probes of length six, then itcontains 4096 (=46) probes. In another aspect, first and second sets ofoligonucleotide probes comprise probes having selected nucleotidesequences designed to detect selected sets of target polynucleotides.Sequences are determined by hybridizing one probe or pool of probe,hybridizing a second probe or a second pool of probes, ligating probesthat form perfectly matched duplexes on their target sequences,identifying those probes that are ligated to obtain sequence informationabout the target sequence, repeating the steps until all the probes orpools of probes have been hybridized, and determining the nucleotidesequence of the target from the sequence information accumulated duringthe hybridization and identification steps.

For sequencing operation, in some embodiments, the sets may be dividedinto subsets that are used together in pools, as disclosed in U.S. Pat.No. 6,864,052, which is incorporated by reference. Probes from the firstand second sets may be hybridized to target sequences either together orin sequence, either as entire sets or as subsets, or pools. In oneaspect, lengths of the probes in the first or second sets are in therange of from 5 to 10 nucleotides, and in another aspect, in the rangeof from 5 to 7 nucleotides, so that when ligated they form ligationproducts with a length in the range of from 10 to 20, and from 10 to 14,respectively.

Kits of the Invention

In the commercialization of the methods described herein, certain kitsfor DNA fragmentation, aliqoting, amplification, construction of randomarrays, and for using the same for various applications are particularlyuseful. Kits for applications of random arrays of the invention include,but are not limited to, kits for determining the nucleotide sequence ofa target polynucleotide, kits for large-scale identification ofdifferences between reference DNA sequences and test DNA sequences, kitsfor profiling exons, and the like. A kit typically comprises at leastone support having a surface and one or more reagents necessary oruseful for constructing a random array of the invention or for carryingout an application therewith. Such reagents include, without limitation,nucleic acid primers, probes, adaptors, enzymes, and the like, and areeach packaged in a container, such as, without limitation, a vial, tubeor bottle, in a package suitable for commercial distribution, such as,without limitation, a box, a sealed pouch, a blister pack and a carton.The package typically contains a label or packaging insert indicatingthe uses of the packaged materials. As used herein, “packagingmaterials” includes any article used in the packaging for distributionof reagents in a kit, including without limitation containers, vials,tubes, bottles, pouches, blister packaging, labels, tags, instructionsheets and package inserts.

In one aspect, kits of the invention for carrying out multiple levels ofrandom fragmentation and aliquoting comprise the following components:(a) mechanical appliance and/or one or more nucleases for generatingrandom fragments from one or more target polynucleotides, (b)amplification reagents for replicating random fragments, and (c) vesselsfor holding aliquots of random fragments. In one embodiment, suchvessels are selected from 96-well plates, 384-well plates, or 1536-wellplates. In another aspect, the enzyme is DNAse I. In yet anotherembodiment, the mechanical appliance is an ultrasonic generator.

Analysis of cDNAs

A human cell contains about 300,000 mRNA molecules with average sizeabout 3 kb, e.g. about one Gb or 16% of the size of all chromosomal DNAin the cell. These mRNAs usually represent on the order of about 10,000different genes. Usually about 50% of all mRNA molecules are generatedfrom about a few hundred highly expressed genes. Large majority of theexpressed genes in a cell are represented with 1-30 mRNA molecules. Fulllength or long cDNA may be prepared fusing mRNA templates. Variousnormalization or cleaning methods may be used to remove majority ofhighly redundant mRNA molecules. Furthermore it is found that at least50% of genes has on average three splice variants. For complete geneexpression analysis there is a need to determine complete or almostcomplete sequences of all different mRNA/cDNA present in the cell. Suchanalysis will give full characterization of the expressed splicevariants and protein haplotypes including expression level of eachvariant. A variant of aliquoting process provides ability to obtain thiscomplete analysis of expressed gene sequences.

For most of the genes that have 1-30 mRNA molecules per cell dilutionand or direct aliquoting of full length or very long cDNA would beadjusted similarly to analysis of 15× coverage of diploid chromosomalDNA. About 96-384 or up to 1536 aliquots assures that 30 or even 100cDNA molecules transcribed from the same gene in most case will beplaced in separate aliquots. Only rarely a aliquot will have two cDNArepresenting the same gene. The number of aliquots may be increased thatmost of the distinct mRNA variants are represented at least two times inthe set of the aliquots or at least 10-20 times for the purpose ofaccurate determination of expression level of each variant. At the sametime highly expressed genes with several hundreds or a few thousands ofcDNAs, if they are not removed, would have multiple cDNA representativesper aliquot. For these genes a special set of 96 or 384 or more lowcomplexity aliquots may be prepared by using about 10-100 fold less cDNAper aliquot.

cDNA in the aliquots may be protected by various carriers such as tRNAor glycogen. cDNA will be replicated 10-1000 fold as full length or inmultiple segments covering the entire cDNA. The obtained DNA may befurther fragmented to second-sized fragments that are appropriate forthe method of choice for sequence analysis. In the preferred methodsecond sized fragments are tagged with an oligonucleotide of knownsequence. A different oligo with one or more base differences is usedfor each aliquot. After tagging DNA from some or all aliquots is mixedto simplify further preparation for sequencing. Sequencing may be donewith various methods as described for chromosomal DNA. In the preferredmode 5-10 fold sequence coverage is obtained for each cDNA in eachaliquot, but as low as 1-2 fold coverage may be used. After sequencing,short reads coming from the same aliquot may be assembled to form fulllength sequences of each of cDNA molecules present in the aliquot.Because in most cases a gene is represented with only one cDNA moleculeper aliquot the actual sequence of that molecule is obtained revelingactual protein haplotype or splice variant that would be synthesizedfrom that mRNA molecule. This is equivalent to full length sequencing ofindividual full-length cDNA clones. By comparing sequence reads orassembled cDNA sequences across aliquots all identical variants may becounted and the number of occurrence indicate expression level for eachmRNA variant.

Traditional methods of cloning have several drawbacks including thepropensity of bacteria to exclude sequences from plasmid replication andthe time consuming and reagent-intensive protocols required to generateclones of individual cDNA molecules. We have previously demonstrated theability to create linear single-stranded amplifications of DNA moleculesthat have been closed into a circular form. These large concatemeric,linear forms arise from a single molecule and can act as efficient,isolated targets for PCR when separated into a single reaction chamber,in much the same way a bacterial colony is picked to retrieve the cDNAcontaining plasmid. We plan to develop this approach as a means toselect cDNA clones without having to pass through a cell-based clonalselection step.

The first step of this procedure will involve ligating a gene specificoligonucleotide directed to the 5-prime end with a poly dA sequence forbinding to the poly dT sequence of the 3-prime end of the cDNA. Thisoligonucleotide acts as a bridge to allow T4 DNA ligase to ligate thetwo ends and form a circle.

The second step of the reaction is to use a primer, or the bridgingoligonucleotide, for a strand displacing polymerase such as Phi 29polymerase to create a concatemer of the circle. The long linearmolecules will then be diluted and arrayed in 1536 well plates such thatwells with single molecules can be selected. To ensure about 10% of thewells contain 1 molecule approximately 90% would have to be sacrificedas having no molecules. To detect the wells that are positive we plan tohybridize a dendrimer that recognizes a universal sequence in the targetto generate 10K-100K dye molecules per molecule of target. Excessdendrimer could be removed through hybridization to biotinylated captureoligos. The wells will be analyzed with a fluorescent plate reader andthe presence of DNA scored. Positive wells will then be re-arrayed toconsolidate the clones into plates with complete wells for furtheramplification

Splice Variant Detection and Exon Profiling

The process described is based on random DNA arrays and “smart” probepools for the identification and quantification of expression levels ofthousands of genes and their splice variants. In eukaryotes, as theprimary transcript emerges from the transcription complex, spliceosomesinteract with splice sites on the primary transcript to excise out theintrons, e.g. Maniatis et al, Nature, 418: 236-243 (2002). However,because of either mutations that alter the splice site sequences, orexternal factors that affect spliceosome interaction with splice sites,alternative splice sites, or cryptic splice sites, could be selectedresulting in expression of protein variants encoded by mRNA withdifferent sets of exons. Surveys of cDNA sequences from large scale ESTsequencing projects indicated that over 50% of the genes have knownsplice variants. In a recent study using a microarray-based approach, itwas estimated that as high as 75% of genes are alternatively spliced,e.g. Johnson et al, Science, 302: 2141-2144 (2003).

The diversity of proteins generated through alternative splicing couldpartially contribute to the complexity of biological processes in highereukaryotes. This also leads to the implication that the aberrantexpression of variant protein forms could be responsible forpathogenesis of diseases. Indeed, alternative splicing has been found toassociate with various diseases like growth hormone deficiency,Parkinson's disease, cystic fibrosis and myotonic dystrophy, e.g.Garcia-Blanco et al, Nature Biotechnology, 22: 535-546 (2004). Becauseof the difficulty in isolating and characterizing novel splice variants,the evidence implicating roles of splice variants in cancer couldrepresent the tip of the iceberg. With the availability of tools thatcould rapidly and reliably characterize splicing patterns of mRNA, itwould help to elucidate the role of alternative splicing in cancer andin disease development in general.

In one aspect, methods of the invention permit large-scale measurementof splice variants with the following steps: (a) Prepare full lengthfirst strand cDNA for targeted or all mRNAs. (b) Circularize thegenerated full length (or all) first strand cDNA molecules byincorporating an adapter sequence. (c) By using primer complementary tothe adapter sequence perform rolling circle replication (RCR) of cDNAcircles to form concatemers with over 100 copies of initial cDNA. (d)Prepare random arrays by attaching RCR produced “cDNA balls” to glasssurface coated with capture oligonucleotide complementary to a portionof the adapter sequence; with an advanced submicron patterned surfaceone mm² can have between 1-10 million cDNA spots; note that theattachment is a molecular process and does not require robotic spottingof individual “cDNA balls” or concatemers. (e) Starting from pre-madeuniversal libraries of 4096 6-mers and 1024 labeled 5-mers, use asophisticated computer program and a simple robotic pipettor to create40-80 pools of about 200 6-mers and 20 5-mers for testing all 10,000 ormore exons in targeted 1000 or more up to all known genes in the sampleorganism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probepools in 40-80 cycles on the same random array using an automatedmicroscope-like instrument with a sensitive 10-mega pixel CCD detectorfor generating an array image for each cycle. (g) Use a computer programto perform spot signal intensity analysis to identify which cDNA is onwhich spot, and if any of the expected exons is missing in any of theanalyzed genes. Obtain exact expression levels for each splice variantby counting occurrences in the array.

This system provides a complete analysis of the exon pattern on a singletranscript, instead of merely providing information on the ratios ofexon usage or quantification of splicing events over the entirepopulation of transcribed genes using the current expression arrayshybridized with labeled mRNA/cDNA. At the maximum limit of itssensitivity, it allows a detailed analysis down to a single molecule ofa mRNA type present in only one in hundreds of other cells; this wouldprovide unique potentials for early diagnosis of cancer cells. Thecombination of selective cDNA preparation with an “array of randomarrays” in a standard 384-well format and with “smart” pools ofuniversal short probes provides great flexibility in designing assays;for examples, deep analysis of a small number of genes in selectedsamples, or more general analysis in a larger number of samples, oranalysis of a large number of genes in smaller number of samples. Theanalysis provides simultaneously 1) detection of each specific splicevariant, 2) quantification of expression of wild type and alternativelyspliced mRNAs. It can also be used to monitor gross chromosomalalterations based on the detection of gene deletions and genetranslocations by loss of heterozygosity and presence of two sub-sets ofexons from two genes in the same transcript on a single spot on therandom array. The exceptional capacity and informativeness of this assayis coupled with simple sample preparation from very small quantities ofmRNA, fully-automated assay based on all pre-made, validated reagentsincluding libraries of universal labeled and unlabeled probes andprimers/adapters that will be ultimately developed for all human andmodel organism genes. The proposed splice variant profiling process isequivalent to high throughput sequencing of individual full length cDNAclones; rSBH throughput can reach one billion cDNA molecules profiled ina 4-8 hour assay. This system will provide a powerful tool to monitorchanges in expression levels of various splice variants during diseaseemergence and progression. It can enable discovery of novel splicevariants or validate known splice variants to serve as biomarkers tomonitor cancer progression. It can also provide means to furtherunderstanding the roles of alternative splice variants and theirpossible uses as therapeutic targets. Universal nature and flexibilityof this low cost and high throughput assay provides great commercialopportunities for cancer research and diagnostics and in all otherbiomedical areas. This high capacity system is ideal for serviceproviding labs or companies.

Preparation of Templates for In Vitro Transcription. Exon Sequences areCloned into the multiple cloning sites (MCS) of plasmid pBluescript, orlike vector. For the purposes of demonstrating the usefulness of theprobe pools, it is not necessary to clone the contiguous full-lengthsequence, nor to maintain the proper protein coding frame. For genesthat are shorter than 1 kb, PCR products are generated from cDNA usinggene specific oligos for the full length sequence. For longer genes, PCRproducts are generated comprising about 500 bp that corresponding tocontiguous block of exons and ordered the fragments by cloning intoappropriate cloning sites in the MCS of pBluescript. This is also theapproach for cloning the alternative spliced versions, since the desiredvariant might not be present in the cDNA source used for PCR.

The last site of the MCS is used to insert a string of 40 A's tosimulate the polyA tails of cellular mRNA. This is to control for thepossibility that the polyA tail might interfere with the samplepreparation step described below, although it is not expected to be aproblem since a poly-dA tail is incorporated in sample preparation ofgenomic fragments as described. T7 RNA polymerase will be used togenerate the run-off transcripts and the RNA generated will be purifiedwith the standard methods.

Preparation of Samples for Arraying. Because the Probe Pools areDesigned for Specific genes, cDNA is prepared for those specific genesonly. For priming the reverse transcription reactions, gene-specificprimers are used, therefore for 1000 genes, 1000 primers are used. Thelocation of the priming site for the reverse transcription is selectedwith care, since it is not reasonable to expect the synthesis of cDNA >2kb to be of high efficiency. It is quite common that the last exon wouldconsist of the end of the coding sequence and a long 3′ untranslatedregion. In the case of CD44 for example, although the full-length mRNAis about 5.7 kb, the 3′ UTR comprises of 3 kb, while the coding regionis only 2.2 kb. Therefore the logical location of the reversetranscription primer site is usually immediately downstream of the endof the coding sequence. For some splice variants, the alternative exonsare often clustered together as a block to create a region ofvariability. In the case of Tenascin C variants (8.5 kb), the mostcommon isoform has a block of 8 extra exons, and there is evidence tosuggest that there is variability in exon usage in that region. So forTenascin C, the primer will be located just downstream of that region.Because of the concern of synthesizing cDNA with length >2 kb, for longgenes, it might be necessary to divide the exons into blocks of 2 kbwith multiple primers.

Reverse transcription reactions may be carried out with commercialsystems, e.g. SuperScript III system from Invitrogen (Carlsbad, Calif.)and the StrataScript system from Stratagene (La Jolla, Calif.). Oncesingle stranded cDNA molecules are produced, the rest of the proceduresinvolved putting on the adaptor sequence, circularization of themolecule and RCR as described above. The 5′ ends of the cDNAs arebasically the incorporated gene-specific primers used for initiating thereverse transcription. By incorporating a 7 base universal tag on the 5′end of the reverse-transcription priming oligos, all the cDNA generatedwill carry the same 7 base sequence at the 5′ end. Thus a singletemplate oligonucleotide that is complementary to both the adaptorsequence and the universal tag can be used to ligate the adaptor to allthe target molecules, without using the template oligonucleotide withdegenerate bases. As for the 3′ end of the cDNA (5′ end of the mRNA)which is usually ill-defined, it may be treated like a random sequenceend of a genomic fragment. Similar methods of adding a polyA tail willbe applied, thus the same circle closing reaction may also be used.

Reverse transcriptases are prone to terminate prematurely to createtruncated cDNAs. Severely truncated cDNAs probably will not have enoughprobe binding sites to be identified with a gene assignment, thus wouldnot be analyzed. cDNA molecules that are close, but not quitefull-length, may show up as splice variant with missing 5′ exons. Ifthere are no corroborating evidence from a sequence database to supportsuch variants, they may be discounted. A way to avoid such problem is toselect for only the full-length cDNA (or those with the desired 3′ end)to be compatible with circle closing reaction, then any truncatedmolecules will not be circularized nor replicated. First adideoxy-cytosine residue can be added to the 3′ end of all the cDNA toblock ligation, then by using a mismatch oligo targeting the desiredsequence, a new 3′ end can be generated by enzyme mismatch cleavageusing T4 endonuclease VII. With the new 3′ end, the cDNA can proceedwith the adding a poly-dA tail and with the standard protocols ofcircularization and replication.

Replicated and arrayed concatemers of the exon fragments may be carriedout using combinatorial SBH, as described above. The algorithm of thefollowing steps may be used to select 5-mer and 6-mer probes for use inthe technique:

Step 1: Select 1000-2000 shortest exons (total about 20-50 kb), and findout matching sequences for each of 1024 available labeled 5-mers. Onaverage each 5-mer will occur 20 times over 20 kb, but some may occurover 50 or over 100 times. By selecting the most frequent 5-mer, thelargest number of short exons will be detected with the single labeledprobe. A goal would be to detect about 50-100 short exons (10%-20% of500 exons) per cycle. Thus less than 10 labeled probes and 50-100unlabeled 6-mers would be sufficient. Small number of labeled probes isfavorable because it minimizes overall fluorescent background.

Step 2. Find out all 6-mers that are contiguous with all sites in all1000 genes that are complementary to 10 selected 5-mers. On average 20such sites will exist in each 2 kb gene. Total number of sites would beabout 20,000, e.g., each 6-mer on average will occur 5 times. Sort6-mers by the hit frequency. The most frequent may have over 20 hits,e.g. such 6-mer will detect 20 genes through combinations with 10labeled probes. Thus, to get a single probe pair for each of the 500genes a minimum of 25 6-mer probes would be required. Realistically, 100to 200 6-mers may be required.

Due to benefits of combinatorial SBH that uses pre-made libraries of6-mer and 5-mer probes 40 probe pools are readily prepared with about200 probes per pool using established pipetting robotics. Theinformation generated is equivalent to having over 3 probes per exon,therefore the use of 8000 5-mers and 6-mers effectively replaces the30,000 longer exons specific probes required for a single set of 1000genes.

Exon profiling. The profiling of exons can be performed in two phases:the gene identification phase and the exon identification phase. In thegene identification phase, each concatemer on the array can be uniquelyidentified with a particular gene. In theory, 10 probe pools orhybridization cycles will be enough to identify 1000 genes using thefollowing scheme. Each gene is assigned a unique binary code. The numberof binary digits thus depends on the total number of genes: 3 digits for8 genes, 10 digits for 1024 genes. Each probe pool is designed tocorrespond to a digit of the binary code and would contain probes thatwould hit a unique combination of half of the genes and one hit per geneonly. Thus for each hybridization cycle, an unique half of the geneswill score a 1 for that digit and the other half will score zero. Tenhybridization cycles with 10 probe pools will generate 1024 uniquebinary codes, enough to assign 1000 unique genes to all the concatemerson the array. To provide redundancy in the identification data, 15-20cycles would be used. If 20 cycles are used, it would provide 1 millionunique binary codes and there should be enough information to accountfor loss of signals due to missing exons or gene deletions. It will alsobe equivalent to having 10 data points per gene (20 cycles of 500 datapoint each give 10,000 data points total), or one positive probe-pairper exon, on average. At this point after 20 cycles, this system iscapable of making assignment of 1 million unique gene identities to theampliots. Therefore by counting gene identities of the ampliots, one candetermine quantitatively the expression level of all the genes (but notsub-typing of splice variants) in any given samples.

After identifying each ampliot with a gene assignment, its exon patternwill be profiled in the exon identification phase. For the exonidentification phase, one exon per gene in all or most of the genes istested per hybridization cycle. In most cases 10-20 exon identificationcycles should be sufficient. Thus, in the case of using 20 exonidentification cycles we will obtain information of 2 probes per each of10 exons in each gene. For genes with more than 20 exons, methods can bedeveloped so that 2 exons per gene can be probed at the same cycle. Onepossibility is using multiple fluorophores of different colors, andanother possibility is to exploit differential hybrid stabilities ofdifferent ligation probe pairs.

In conclusion, a total of about 40 assay cycles will provide sufficientinformation to obtain gene identity at each spot and to provide threematching probe-pairs for each of 10,000 exons with enough informationalredundancy to provide accurate identification of missing exons due toalternative splicing or chromosomal deletions.

DEFINITIONS

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g. Komberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like.

“Amplicon” means the product of a polynucleotide amplification reaction.That is, it is a population of polynucleotides, usually double stranded,that are replicated from one or more starting sequences. The one or morestarting sequences may be one or more copies of the same sequence, or itmay be a mixture of different sequences. Amplicons may be produced by avariety of amplification reactions whose products are multiplereplicates of one or more target nucleic acids. Generally, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, have complementsin a template polynucleotide that are required for the creation ofreaction products. In one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerase chain reactions (PCRs), linear polymerase reactions,nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S.Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al,U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No. 5,399,491(“NASBA”); Lizardi, U.S. Pat. No. 5,854,033; Aono et al, Japanese patentpubl. JP 4-262799 (rolling circle amplification); and the like. In oneaspect, amplicons of the invention are produced by PCRs. Anamplification reaction may be a “real-time” amplification if a detectionchemistry is available that permits a reaction product to be measured asthe amplification reaction progresses, e.g. “real-time PCR” describedbelow, or “real-time NASBA” as described in Leone et al, Nucleic AcidsResearch, 26: 2150-2155 (1998), and like references. As used herein, theterm “amplifying” means performing an amplification reaction. A“reaction mixture” means a solution containing all the necessaryreactants for performing a reaction, which may include, but not belimited to, buffering agents to maintain pH at a selected level during areaction, salts, co-factors, scavengers, and the like.

“Complementary or substantially complementary” refers to thehybridization or base pairing or the formation of a duplex betweennucleotides or nucleic acids, such as, for instance, between the twostrands of a double stranded DNA molecule or between an oligonucleotideprimer and a primer binding site on a single stranded nucleic acid.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 80% of the nucleotides of the other strand, usuallyat least about 90% to 95%, and more preferably from about 98 to 100%.Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25nucleotides, preferably at least about 75%, more preferably at leastabout 90% complementary. See, M. Kanehisa Nucleic Acids Res. 12:203(1984), incorporated herein by reference.

“Duplex” means at least two oligonucleotides and/or polynucleotides thatare fully or partially complementary undergo Watson-Crick type basepairing among all or most of their nucleotides so that a stable complexis formed. The terms “annealing” and “hybridization” are usedinterchangeably to mean the formation of a stable duplex. “Perfectlymatched” in reference to a duplex means that the poly- oroligonucleotide strands making up the duplex form a double strandedstructure with one another such that every nucleotide in each strandundergoes Watson-Crick basepairing with a nucleotide in the otherstrand. The term “duplex” comprehends the pairing of nucleoside analogs,such as deoxyinosine, nucleosides with 2-aminopurine bases, PNAs, andthe like, that may be employed. A “mismatch” in a duplex between twooligonucleotides or polynucleotides means that a pair of nucleotides inthe duplex fails to undergo Watson-Crick bonding.

“Genetic locus,” or “locus” in reference to a genome or targetpolynucleotide, means a contiguous subregion or segment of the genome ortarget polynucleotide. As used herein, genetic locus, or locus, mayrefer to the position of a nucleotide, a gene, or a portion of a gene ina genome, including mitochondrial DNA, or it may refer to any contiguousportion of genomic sequence whether or not it is within, or associatedwith, a gene. In one aspect, a genetic locus refers to any portion ofgenomic sequence, including mitochondrial DNA, from a single nucleotideto a segment of few hundred nucleotides, e.g. 100-300, in length.

“Genetic variant” means a substitution, inversion, insertion, ordeletion of one or more nucleotides at genetic locus, or a translocationof DNA from one genetic locus to another genetic locus. In one aspect,genetic variant means an alternative nucleotide sequence at a geneticlocus that may be present in a population of individuals and thatincludes nucleotide substitutions, insertions, and deletions withrespect to other members of the population. In another aspect,insertions or deletions at a genetic locus comprises the addition or theabsence of from 1 to 10 nucleotides at such locus, in comparison withthe same locus in another individual of a population.

“Hybridization” refers to the process in which two single-strandedpolynucleotides bind non-covalently to form a stable double-strandedpolynucleotide. The term “hybridization” may also refer totriple-stranded hybridization. The resulting (usually) double-strandedpolynucleotide is a “hybrid” or “duplex.” “Hybridization conditions”will typically include salt concentrations of less than about 1M, moreusually less than about 500 mM and less than about 200 mM. A“hybridization buffer” is a buffered salt solution such as 5×SSPE, orthe like. Hybridization temperatures can be as low as 5° C., but aretypically greater than 22° C., more typically greater than about 30° C.,and preferably in excess of about 37° C. Hybridizations are usuallyperformed under stringent conditions, i.e. conditions under which aprobe will hybridize to its target subsequence. Stringent conditions aresequence-dependent and are different in different circumstances. Longerfragments may require higher hybridization temperatures for specifichybridization. As other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one alone. Generally, stringent conditions areselected to be about 5° C. lower than the T_(m) for the specificsequence at s defined ionic strength and pH. Exemplary stringentconditions include salt concentration of at least 0.01 M to no more than1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and atemperature of at least 25° C. For example, conditions of 5×SSPE (750 mMNaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C. are suitable for allele-specific probe hybridizations. For stringentconditions, see for example, Sambrook, Fritsche and Maniatis. “MolecularCloning A laboratory Manual” 2^(nd) Ed. Cold Spring Harbor Press (1989)and Anderson “Nucleic Acid Hybridization” 1^(st) Ed., BIOS ScientificPublishers Limited (1999), which are hereby incorporated by reference inits entirety for all purposes above. “Hybridizing specifically to” or“specifically hybridizing to” or like expressions refer to the binding,duplexing, or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA.

“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g. oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon of a terminal nucleotide of one oligonucleotide with 3′ carbon ofanother oligonucleotide. A variety of template-driven ligation reactionsare described in the following references, which are incorporated byreference: Whitely et al, U.S. Pat. No. 4,883,750; Letsinger et al, U.S.Pat. No. 5,476,930; Fung et al, U.S. Pat. No. 5,593,826; Kool, U.S. Pat.No. 5,426,180; Landegren et al, U.S. Pat. No. 5,871,921; Xu and Kool,Nucleic Acids Research, 27: 875-881 (1999); Higgins et al, Methods inEnzymology, 68: 50-71 (1979); Engler et al, The Enzymes, 15: 3-29(1982); and Namsaraev, U.S. patent publication 2004/0110213. Enzymaticligation usually takes place in a ligase buffer, which is a bufferedsalt solution containing any required divalent cations, cofactors, andthe like, for the particular ligase employed.

“Microarray” or “array” refers to a solid phase support having asurface, usually planar or substantially planar, which carries an arrayof sites containing nucleic acids, such that each member site of thearray comprises identical copies of immobilized oligonucleotides orpolynucleotides and is spatially defined and not overlapping with othermember sites of the array; that is, the sites are spatially discrete. Insome cases, sites of a microarray may also be spaced apart as well asdiscrete; that is, different sites do not share boundaries, but areseparated by inter-site regions, usually free of bound nucleic acids.Spatially defined hybridization sites may additionally be “addressable”in that its location and the identity of its immobilized oligonucleotideare known or predetermined, for example, prior to its use. In someaspects, the oligonucleotides or polynucleotides are single stranded andare covalently attached to the solid phase support, usually by a 5′-endor a 3′-end. In other aspects, oligonucleotides or polynucleotides areattached to the solid phase support non-covalently, e.g. by abiotin-streptavidin linkage, hybridization to a capture oligonucleotidethat is covalently bound, and the like. Conventional microarraytechnology is reviewed in the following references: Schena, Editor,Microarrays: A Practical Approach (IRL Press, Oxford, 2000); Southern,Current Opin. Chem. Biol., 2: 404-410 (1998); Nature GeneticsSupplement, 21: 1-60 (1999). As used herein, “random array” or “randommicroarray” refers to a microarray whose spatially discrete regions ofoligonucleotides or polynucleotides are not spatially addressed. Thatis, the identity of the attached oligonucleoties or polynucleotides isnot discernable, at least initially, from its location, but may bedetermined by a particular operation on the array, e.g. sequencing,hybridizing decoding probes, or the like. Random microarrays arefrequently formed from a planar array of microbeads, e.g. Brenner et al,Nature Biotechnology, 18: 630-634 (2000); Tulley et al, U.S. Pat. No.6,133,043; Stuelpnagel et al, U.S. Pat. No. 6,396,995; Chee et al, U.S.Pat. No. 6,544,732; and the like.

“Mismatch” means a base pair between any two of the bases A, T (or U forRNA), G, and C other than the Watson-Crick base pairs G-C and A-T. Theeight possible mismatches are A-A, T-T, G-G, C-C, T-G, C-A, T-C, andA-G.

“Mutation” and “polymorphism” are usually used somewhat interchangeablyto mean a DNA molecule, such as a gene, which differs in nucleotidesequence from a reference DNA sequence, or wild type sequence, or normaltissue sequence, by one or more bases, insertions, and/or deletions. Insome contexts, the usage of Cotton (Mutation Detection, OxfordUniversity Press, Oxford, 1997) is followed in that a mutation isunderstood to be any base change whether pathological to an organism ornot, whereas a polymorphism is usually understood to be a base changewith no direct pathological consequences.

“Nucleoside” as used herein includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). “Analogs” inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described by Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,Chemical Reviews, 90: 543-584 (1990), or the like, with the proviso thatthey are capable of specific hybridization. Such analogs includesynthetic nucleosides designed to enhance binding properties, reducecomplexity, increase specificity, and the like. Polynucleotidescomprising analogs with enhanced hybridization or nuclease resistanceproperties are described in Uhlman and Peyman (cited above); Crooke etal, Exp. Opin. Ther. Patents, 6: 855-870 (1996); Mesmaeker et al,Current Opinion in Structural Biology, 5: 343-355 (1995); and the like.Exemplary types of polynucleotides that are capable of enhancing duplexstability include oligonucleotide N3′→P5′ phosphoramidates (referred toherein as “amidates”), peptide nucleic acids (referred to herein as“PNAs”), oligo-2′-O-alkylribonucleotides, polynucleotides containing C-5propynylpyrimidines, locked nucleic acids (LNAs), and like compounds.Such oligonucleotides are either available commercially or may besynthesized using methods described in the literature.

“Oligonucleotide tag” means an oligonucleotide that is attached to apolynucleotide and is used to identify and/or track the polynucleotidein a reaction. Usually, a oligonucleotide tag is attached to the 3′- or5′-end of a polynucleotide to form a linear conjugate, sometime referredto herein as a “tagged polynucleotide,” or equivalently, an“oligonucleotide tag-polynucleotide conjugate,” or “tag-polynucleotideconjugate.” Oligonucleotide tags may vary widely in size andcompositions; the following references provide guidance for selectingsets of oligonucleotide tags appropriate for particular embodiments:Brenner, U.S. Pat. No. 5,635,400; Brenner et al, Proc. Natl. Acad. Sci.,97: 1665-1670 (2000); Church et al, European patent publication 0 303459; Shoemaker et al, Nature Genetics, 14: 450-456 (1996); Morris et al,European patent publication 0799897A1; Wallace, U.S. Pat. No. 5,981,179;and the like. Lengths of oligonucleotide tags can vary widely, and theselection of a particular lengths depend on several factors including,without limitation, whether the oligonucleotide tags are employedprimarily in hybridization reactions or primarily in enzymaticreactions, whether they are labeled, whether such labeling is direct orindirect, the number of distinguishable oligonucleotide tags required,and the like. In one aspect, oligonucleotide tags can each have a lengthwithin a range of from 2 to 36 nucleotides, or from 4 to 30 nucleotides,or from 8 to 20 nucleotides, respectively. In one aspect,oligonucleotide tags are used in sets, or repertoires, wherein eacholigonucleotide tag of the set has a unique nucleotide sequence. In someembodiments, particularly where oligonucleotide tags are used to sortpolynucleotides, or where they are identified by specific hybridization,each oligonucleotide tag of such a set has a melting temperature that issubstantially the same as that of every other member of the same set. Insuch aspects, the melting temperatures of oligonucleotide tags within aset are within 110° C. of one another; in another embodiment, they arewithin 5° C. of one another; and in another embodiment, they are within2° C. of one another. A set of oligonucleotide tags may have a size inthe range of from several tens to many thousands, or even millions, e.g.50 to 1.6×10⁶. In another embodiment, such a size is in the range offrom 200 to 40,000; or from 1000 to 40,000; or from 1000 to 10,000.Where oligonucleotide tags are used to label or identify fragments froma particular organism or species whose genome sequence is known, tagsequences may be selected to be distinguishable from the genomicsequences of such organism or species.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in aconventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C. The term “PCR” encompasses derivativeforms of the reaction, including but not limited to, RT-PCR, real-timePCR, nested PCR, quantitative PCR, multiplexed PCR, and the like.Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to afew hundred μL, e.g. 200 μL. “Reverse transcription PCR,” or “RT-PCR,”means a PCR that is preceded by a reverse transcription reaction thatconverts a target RNA to a complementary single stranded DNA, which isthen amplified, e.g. Tecott et al, U.S. Pat. No. 5,168,038, which patentis incorporated herein by reference. “Real-time PCR” means a PCR forwhich the amount of reaction product, i.e. amplicon, is monitored as thereaction proceeds. There are many forms of real-time PCR that differmainly in the detection chemistries used for monitoring the reactionproduct, e.g. Gelfand et al, U.S. Pat. No. 5,210,015 (“taqman”); Wittweret al, U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes);Tyagi et al, U.S. Pat. No. 5,925,517 (molecular beacons); which patentsare incorporated herein by reference. Detection chemistries forreal-time PCR are reviewed in Mackay et al, Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference.“Nested PCR” means a two-stage PCR wherein the amplicon of a first PCRbecomes the sample for a second PCR using a new set of primers, at leastone of which binds to an interior location of the first amplicon. Asused herein, “initial primers” in reference to a nested amplificationreaction mean the primers used to generate a first amplicon, and“secondary primers” mean the one or more primers used to generate asecond, or nested, amplicon. “Multiplexed PCR” means a PCR whereinmultiple target sequences (or a single target sequence and one or morereference sequences) are simultaneously carried out in the same reactionmixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228(1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified. “Quantitative PCR” means aPCR designed to measure the abundance of one or more specific targetsequences in a sample or specimen. Quantitative PCR includes bothabsolute quantitation and relative quantitation of such targetsequences. Quantitative measurements are made using one or morereference sequences that may be assayed separately or together with atarget sequence. The reference sequence may be endogenous or exogenousto a sample or specimen, and in the latter case, may comprise one ormore competitor templates. Typical endogenous reference sequencesinclude segments of transcripts of the following genes: β-actin, GAPDH,β₂-microglobulin, ribosomal RNA, and the like. Techniques forquantitative PCR are well-known to those of ordinary skill in the art,as exemplified in the following references that are incorporated byreference: Freeman et al, Biotechniques, 26: 112-126 (1999);Becker-Andre et al, Nucleic Acids Research, 17: 9437-9447 (1989);Zimmerman et al, Biotechniques, 21: 268-279 (1996); Diviacco et al,Gene, 122: 3013-3020 (1992); Becker-Andre et al, Nucleic Acids Research,17: 9437-9446 (1989); and the like.

“Polynucleotide” or “oligonucleotide” are used interchangeably and eachmean a linear polymer of nucleotide monomers. As used herein, the termsmay also refer to double stranded forms. Monomers making uppolynucleotides and oligonucleotides are capable of specifically bindingto a natural polynucleotide by way of a regular pattern ofmonomer-to-monomer interactions, such as Watson-Crick type of basepairing, base stacking, Hoogsteen or reverse Hoogsteen types of basepairing, or the like, to form duplex or triplex forms. Such monomers andtheir internucleosidic linkages may be naturally occurring or may beanalogs thereof, e.g. naturally occurring or non-naturally occurringanalogs. Non-naturally occurring analogs may include PNAs,phosphorothioate internucleosidic linkages, bases containing linkinggroups permitting the attachment of labels, such as fluorophores, orhaptens, and the like. Whenever the use of an oligonucleotide orpolynucleotide requires enzymatic processing, such as extension by apolymerase, ligation by a ligase, or the like, one of ordinary skillwould understand that oligonucleotides or polynucleotides in thoseinstances would not contain certain analogs of internucleosidiclinkages, sugar moities, or bases at any or some positions, when suchanalogs are incompatable with enzymatic reactions. Polynucleotidestypically range in size from a few monomeric units, e.g. 5-40, when theyare usually referred to as “oligonucleotides,” to several thousandmonomeric units. Whenever a polynucleotide or oligonucleotide isrepresented by a sequence of letters (upper or lower case), such as“ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine,“I” denotes deoxyinosine, “U” denotes uridine, unless otherwiseindicated or obvious from context. Unless otherwise noted theterminology and atom numbering conventions will follow those disclosedin Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York,1999). Usually polynucleotides comprise the four natural nucleosides(e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine forDNA or their ribose counterparts for RNA) linked by phosphodiesterlinkages; however, they may also comprise non-natural nucleotideanalogs, e.g. including modified bases, sugars, or internucleosidiclinkages. It is clear to those skilled in the art that where an enzymehas specific oligonucleotide or polynucleotide substrate requirementsfor activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,then selection of appropriate composition for the oligonucleotide orpolynucleotide substrates is well within the knowledge of one ofordinary skill, especially with guidance from treatises, such asSambrook et al, Molecular Cloning, Second Edition (Cold Spring HarborLaboratory, New York, 1989), and like references.

“Primer” means an oligonucleotide, either natural or synthetic, that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.The sequence of nucleotides added during the extension process aredetermined by the sequence of the template polynucleotide. Usuallyprimers are extended by a DNA polymerase. Primers usually have a lengthin the range of from 9 to 40 nucleotides, or in some embodiments, from14 to 36 nucleotides.

“Readout” means a parameter, or parameters, which are measured and/ordetected that can be converted to a number or value. In some contexts,readout may refer to an actual numerical representation of suchcollected or recorded data. For example, a readout of fluorescentintensity signals from a microarray is the position and fluorescenceintensity of a signal being generated at each hybridization site of themicroarray; thus, such a readout may be registered or stored in variousways, for example, as an image of the microarray, as a table of numbers,or the like.

“Solid support”, “support”, and “solid phase support” are usedinterchangeably and refer to a material or group of materials having arigid or semi-rigid surface or surfaces. In many embodiments, at leastone surface of the solid support will be substantially flat, although insome embodiments it may be desirable to physically separate synthesisregions for different compounds with, for example, wells, raisedregions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. Microarraysusually comprise at least one planar solid phase support, such as aglass microscope slide.

“Reference sequence” or “reference population” of DNA refers toindividual DNA sequences or a collection of DNAs (or RNAs derived fromit) which is compared to a test population of DNA or RNA, (or “test DNAsequence,” or “test DNA population”) by the formation of heteroduplexesbetween the complementary strands of the reference DNA population andtest DNA population. If perfectly matched heteroduplexes form, then therespective members of the reference and test populations are identical;otherwise, they are variants of one another. Typically, the nucleotidesequences of members of the reference population are known and thesequences typically are listed in sequence databases, such as Genbank,Embl, or the like. In one aspect, a reference population of DNA maycomprise a cDNA library or genomic library from a known cell type ortissue source. For example, a reference population of DNA may comprise acDNA library or a genomic library derived from the tissue of a healthyindividual and a test population of DNA may comprise a cDNA library orgenomic library derived from the same tissue of a diseased individual.Reference populations of DNA may also comprise an assembled collectionof individual polynucleotides, cDNAs, genes, or exons thereof, e.g.genes or exons encoding all or a subset of known p53 variants, genes ofa signal transduction pathway, or the like.

“Specific” or “specificity” in reference to the binding of one moleculeto another molecule, such as a labeled target sequence for a probe,means the recognition, contact, and formation of a stable complexbetween the two molecules, together with substantially less recognition,contact, or complex formation of that molecule with other molecules. Inone aspect, “specific” in reference to the binding of a first moleculeto a second molecule means that to the extent the first moleculerecognizes and forms a complex with another molecules in a reaction orsample, it forms the largest number of the complexes with the secondmolecule. Preferably, this largest number is at least fifty percent.Generally, molecules involved in a specific binding event have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the molecules binding to each other. Examples of specificbinding include antibody-antigen interactions, enzyme-substrateinteractions, formation of duplexes or triplexes among polynucleotidesand/or oligonucleotides, receptor-ligand interactions, and the like. Asused herein, “contact” in reference to specificity or specific bindingmeans two molecules are close enough that weak noncovalent chemicalinteractions, such as Van der Waal forces, hydrogen bonding,base-stacking interactions, ionic and hydrophobic interactions, and thelike, dominate the interaction of the molecules.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theTm of nucleic acids are well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation. Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other references(e.g., Allawi, H. T. & SantaLucia, J., Jr., Biochemistry 36, 10581-94(1997)) include alternative methods of computation which take structuraland environmental, as well as sequence characteristics into account forthe calculation of Tm.

“Sample” usually means a quantity of material from a biological,environmental, medical, or patient source in which detection,measurement, or labeling of target nucleic acids is sought. On the onehand it is meant to include a specimen or culture (e.g., microbiologicalcultures). On the other hand, it is meant to include both biological andenvironmental samples. A sample may include a specimen of syntheticorigin. Biological samples may be animal, including human, fluid, solid(e.g., stool) or tissue, as well as liquid and solid food and feedproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Biological samples may include materials takenfrom a patient including, but not limited to cultures, blood, saliva,cerebral spinal fluid, pleural fluid, milk, lymph, sputum, semen, needleaspirates, and the like. Biological samples may be obtained from all ofthe various families of domestic animals, as well as feral or wildanimals, including, but not limited to, such animals as ungulates, bear,fish, rodents, etc. Environmental samples include environmental materialsuch as surface matter, soil, water and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

The above teachings are intended to illustrate the invention and do notby their details limit the scope of the claims of the invention. Whilepreferred illustrative embodiments of the present invention aredescribed, it will be apparent to one skilled in the art that variouschanges and modifications may be made therein without departing from theinvention, and it is intended in the appended claims to cover all suchchanges and modifications that fall within the true spirit and scope ofthe invention.

1. A method of characterizing nucleotide sequences of one or more targetpolynucleotides, said method comprising the steps of: (a) fragmentingsaid one or more target polynucleotides present in a predeterminedcoverage amount to form a population containing overlapping first-sizedfragments having an average length less than those of said targetpolynucleotides; (b) forming a number of separate mixtures from saidpopulation of first-sized fragments, such number being selected suchthat a majority of first-sized fragments in each separate mixture isnon-overlapping with every other first-sized fragment of the samemixture and such that the mixture of origin of each such first-sizedfragment is determinable; (c) generating for each of said separatemixtures a plurality of target concatemers from said first-sizedfragments, each target concatemer comprising multiple copies of at leasta portion of a first-sized fragment; (d) disposing said targetconcatemers on a surface; and (e) generating a number of sequence readsfrom target concatemers of each of said separate mixtures, such sequencereads each having a length less than those of said first-sizedfragments, and such numbers of sequence reads being selected such thatsaid sequence reads of each separate mixture covers a majority offirst-sized fragments therein, and providing complete or partialnucleotide sequences of said one or more target polynucleotides byordering said sequence reads from said target concatemers.
 2. The methodof claim 1 wherein said step of forming includes replicating saidfirst-sized fragments in said separate mixtures.
 3. The method of claim1 further including a step subsequent to said forming step (b) oftagging each of said first-sized fragments with an oligonucleotide tagfor determining said separate mixtures thereof.
 4. The method of claim 3wherein said step of generating a number of sequence reads from saidtarget concatemers includes determining a nucleotide sequence of saidoligonucleotide tags.
 5. The method of claim 1 wherein said step ofgenerating said number of sequence reads comprises: (a) hybridizing oneor more probes from a first set of probes to said target concatemersunder conditions that permit formation of perfectly matched duplexesbetween said one or more probes and complementary sequences on targetconcatemers; (b) hybridizing one or more probes from a second set ofprobes to said target concatemers under conditions that permit formationof perfectly matched duplexes between said one or more probes andcomplementary sequences on target concatemers; (c) ligating probes fromsaid first and second sets hybridized to a target concatemer atcontiguous sites; (d) identifying sequences of said ligated first andsecond probes; and (e) repeating steps (a) through (d) until saidsequences of said first-sized fragments of a majority of said separatemixtures is substantially determined from said sequences of said ligatedprobes.
 6. The method of claim 1 wherein said one or more targetpolynucleotides contain repetitive sequence regions each having a lengthgreater than those of said sequence reads.
 7. The method of claim 1wherein said ordering said sequence information depends on identities ofsaid separate mixtures from which each of said first-sized fragment isderived.
 8. The method of claim 2 wherein said average length of saidfirst-sized fragments is in a range of from 50 to 2000 bases.
 9. Themethod of claim 2 wherein said average length of said first-sizedfragments is in the range of from one third to one thousandth of that ofsaid one or more target polynucleotides.
 10. The method of claim 2wherein at least sixty percent of said first-sized fragments arenon-overlapping in said separate mixtures.
 11. A method ofcharacterizing nucleotide sequences of one or more targetpolynucleotides, said method comprising: (a) fragmenting said one ormore target polynucleotides present in a predetermined coverage amountto form a population containing overlapping first-sized fragments havingan average length less than those of the target polynucleotides; (b)forming a number of separate mixtures from said population offirst-sized fragments, such number being selected such that a majorityof first-sized fragments in said separate mixtures are non-overlappingwith other first-sized fragments of the same mixture; (c) fragmentingsaid first-sized fragments in said mixtures to form a population ofsecond-sized fragments for each mixture such that said second-sizedfragments have an average length less than those of the first-sizedfragments and such that said mixture of origin said second-sizedfragments is determinable; (d) generating for said separate mixtures aplurality of target concatemers from said second-sized fragments, eachtarget concatemer comprising multiple copies of a second-sized fragment;(e) disposing said target concatemers on a surface; (f) generating anumber of sequence reads from target concatemers of each of saidseparate mixtures, such sequence reads each having a length less thanthose of said second-sized fragments, and such numbers of sequence readsbeing selected such that sequence reads of each said separate mixturecovers a majority of second-sized fragments therein, providing completeor partial nucleotide sequences of said one or more targetpolynucleotides by ordering said sequence reads from said targetconcatemers.
 12. The method of claim 11 wherein said step of formingincludes replicating said first-sized fragments in said separatemixtures.
 13. The method of claim 12 wherein said average length of saidfirst-sized fragments is less than 300 kilobases and said average lengthof said second-sized fragments is in a range of from 50 to 600 bases.14. The method of claim 12 wherein said average length of saidfirst-sized fragments is in a range of from one third to one thousandthof that of said one or more target polynucleotides and said averagelength of said second-sized fragments is in a range of from one third toone thousandth of that of said first-sized fragments.
 15. The method ofclaim 12 wherein said second-sized fragments are non-overlapping in atleast sixty-percent of said separate mixtures.
 16. The method of claim12 wherein said one or more target polynucleotides are one or morebacterial genomes.
 17. The method of claim 12 wherein said one or moretarget polynucleotides are haploid strands of a mammalian genome. 18.The method of claim 11, further including a step of tagging each of saidsecond-sized fragments with an oligonucleotide tag for determining saidseparate mixtures thereof.
 19. The method of claim 18 wherein said stepof generating a number of sequence reads from target concatemers of eachof said separate mixtures includes determining a nucleotide sequence ofsaid oligonucleotide tag.
 20. The method of claim 11 wherein said one ormore target polynucleotides contain repetitive sequence regions eachhaving a length greater than that of said nucleotide sequence.
 21. Themethod of claim 12 further including separating said first-sizedfragments by size prior to said step of forming said separate mixtures.22. The method of claim 11 wherein said step of generating said numberof sequence reads comprises: (a) hybridizing one or more probes from afirst set of probes to said target concatemers under conditions thatpermit formation of perfectly matched duplexes between said one or moreprobes and complementary sequences on target concatemers; (b)hybridizing one or more probes from a second set of probes to saidtarget concatemers under conditions that permit formation of perfectlymatched duplexes between said one or more probes and complementarysequences on target concatemers; (c) ligating probes from said first andsecond sets hybridized to a target concatemer at contiguous sites; (d)identifying sequences of said ligated first and second probes; and (e)repeating steps (a) through (d) until sequences of a majority of saidfirst-sized fragments of each of said separate mixtures is determinedfrom identities of said sequences of said ligated probes.
 23. The methodof claim 11 wherein each of said first-sized fragments has anoligonucleotide tag attached to an end, said oligonucleotide tagidentifying said separate mixture of such first-sized fragment.
 24. Amethod of characterizing nucleotide sequences of one or more targetpolynucleotides, said method comprising: (a) fragmenting said one ormore target polynucleotides present in a predetermined coverage amountto form a population containing overlapping first-sized fragments havingan average length substantially less than those of said targetpolynucleotides, said first-sized fragments being initial prior-sizedfragments; (b) generating a set of separate mixtures from eachpopulation of prior-sized fragments, such set being a tier having anumber of mixtures therein, each such number being selected such thatsubstantially every prior-sized fragment in a separate mixture isnon-overlapping with every other prior-sized fragment of the samemixture; (c) fragmenting each of said prior-sized fragments in each ofsaid mixtures to form a population of second-sized fragments for eachmixture such that said second-sized fragments have an average lengthsubstantially less than those of said first-sized fragments and suchthat mixtures of origin of each such second-sized fragment can beidentified, said second-sized fragments being prior-sized fragments of anext step of fragmenting; (d) repeating steps (b) and (c) until a finaltier of mixtures is obtained; (e) disposing said second-sized fragmentson a surface; (f) determining sequence information from at least aportion of one or more second-sized fragments of each mixture in saidfinal tier; and (g) providing complete or partial nucleotide sequencesof said one or more target polynucleotides by ordering the sequenceinformation from said final tier of mixtures, wherein such orderingdepends on identities of mixtures of origin of at least a portion ofsuch sequence information.
 25. A method of characterizing nucleotidesequences of one or more target polynucleotides, said method comprising:(a) forming a plurality of tiers of mixtures that comprise a hierarchyof nested fragments of said one or more target polynucleotides, eachmixture of each prior tier being divided into a number of mixtures in asubsequent tier, at least one tier having mixtures with nonoverlappingfragments, and said plurality of tiers having a final tier whereinmixtures of prior tiers can be identified for each fragment of eachmixture of said final tier; (b) generating for said tiers of mixtures aplurality of target concatemers from said fragments of each final tier,each target concatemer comprising multiple copies of a fragment and eachplurality of target concatemers including a number of fragments thatcovers a majority of said nested fragments of its respective final tier;(c) disposing said target concatemers on a surface; (d) generating anumber of sequence reads from said target concatemers, such sequencereads each having a length less than those of said fragments of saidfinal tier, and each of such numbers of sequence reads being selectedsuch that said sequences reads of each final tier covers a majority ofnested fragments therein, providing complete or partial nucleotidesequences of said one or more target polynucleotides by ordering thesequence information from said final tier of mixtures.
 26. The method ofclaim 25 wherein said step of forming includes replicating saidfragments of each of said mixtures of each of said subsequent tiers. 27.The method of 26 wherein said ordering said sequence information dependson identities of said mixtures at each said tier from which each saidfragment in said final tier is derived.
 28. The method of claim 26wherein fragments are non-overlapping in at least sixty-percent of saidtiers of mixtures.
 29. The method of claim 25 further including a stepof tagging each of said fragments in each of said tiers with anoligonucleotide tag to identify each of said separate mixtures of eachof said tiers.
 30. The method of claim 29 wherein said step ofgenerating a number of sequence reads from target concatemers includesdetermining a nucleotide sequence of each of said oligonucleotide tags.31. The method of claim 26 wherein said one or more targetpolynucleotides contain repetitive sequence regions each having a lengthgreater than that of said nucleotide sequence.
 32. The method of claim1, 11, or 25 wherein said target concatemers are disposed on saidsurface at a density of at least 1000 per μm².
 33. The method of claim1, 11 or 25 wherein said ordering said sequence reads from said targetconcatemers depends on the mixture of origin of at least a portion ofsaid sequence information.